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COLUMBIA    UNIVERSITY 
EDWARD  G.  JANEWAY 
MEMORIAL  LIBRARY 


Digitized  by  the  Internet  Archive 

in  2010  with  funding  from 

Open  Knowledge  Commons 


http://www.archive.org/details/reportofmedicalcOOmedi 


Report  of 

The  Medical  Commission  for  the 
Investigation  of  Acute  Respiratory 
Diseases  of  the  Department  of 
Health  of  the   City   of  New   York 


Part  I 
Studies  on  the   Pneumococcus 


Reprinted  from 

The  Journal  of  Experimental  Medicine,  1905 

Vol.  VII,  No.  5 


The    Knickerbocker    Press 

(G.    p.    Putnam's    Sons) 

N  Ew  York 


CONTENTS 


Preface       ...........        v 

Introductory  Note  to  Studies  on  the  Pneumococcus  under  the 
Auspices  of  the  Medical  Commission  for  the  Investigation 
of  Acute  Respiratory  Diseases  of  the  Department  of  Health 
of  the  City  of  New  York     .......        ix 

Park,  William  H.,  and  Williams,  A.  W.  A  Study  of  Pneu- 
mococci :  A  Comparison  between  the  Pneumococci  Found 
in  the  Throat  Secretions  of  Healthy  Persons  Living  in  both 
City  and  Country,  and  those  Obtained  from  Pneumonic 
Exudates  and  Diseased  Mucous  Membranes         .         .         .         1 

Collins,  Katherine  E.     The  Application  of  the  Reaction  of 

Agglutination  to  the  Pneumococcus    .....       18 

LoNGCOPE,  Warfield  T.,  and  Fox,  W.  W.  A  Comparative 
Study  of  Pneumococci  and  Streptococci  from  the  Mouths 
of  Healthy  Individuals  and  from  Pathological  Conditions    .       28 

NoRRis,  Charles,  and  Pappenheimer,  Alwin  M.  A  Study 
of  Pneumococci  and  Allied  Organisms  in  Human  Mouths 
and  Lungs  after  Death         .......       48 

Duval,  Charles  W.,  and  Lewis,  Paul  A.     Studies  on  the 

Pneumococcus     .........       71 

Plate  XXIX. 

Buerger,  Leo.  Studies  of  the  Pneumococcus  and  Allied  Or- 
ganisms with  Reference  to  their  Occurrence  in  the  Human 
Mouth 95 

Hiss,  Philip  Hanson,  Jr.,  assisted  by  Borden,  John  Harvey, 
and  Knapp,  Clinton  Beecham.  A  Comparative  Study 
of  Pneumococci  and  Allied  Organisms      ....     145 


iv  Contents 

Wood,  Francis  Carter.  The  Viability  of  the  Pneumococcus 
after  Drying :  A  Study  of  One  of  the  Factors  in  Pneumonic 
Infection      .         .       ^ 190 

LoNGCOPE,  Warfield  T.     a  Note  upon  the  Growth  of  Pneu- 

mococci  in  Blood  Serum      .......     224 


PREFACE. 

For  some  years  the  problem  presented  by  the  very  large  and 
constantly  increasing  death-rate  from  the  acute  respiratory 
diseases  has  been  the  cause  of  serious  concern  to  the  Board  of 
Health  of  New  York  City,  and  in  August,  1904,  with  the  con- 
sent of  the  Mayor  and  the  Board  of  Estimate,  it  appointed  a 
Medical  Commission  to  conduct  an  investigation  as  to  the  causes 
for  the  great  prevalence  of  the  acute  respiratory  diseases  in  the 
city,  with  the  hope  that  some  means  could  be  devised  for  re- 
ducing the  excessive  morbidity  and  mortality  from  this  cause. 

The  problem  has  long  been  recognized  as  one  of  the  first  in 
sanitary  importance,  and  efforts  have  frequently  been  made  to 
find  some  method  for  its  solution.  The  inherent  difficulties  of 
the  problem,  how^ever,  are  so  great  and  the  other  questions 
pressing  for  consideration  have  been  so  numerous  and  important 
that  no  determined  efforts  have  hitherto  been  made  to  discover 
means  or  devise  methods  to  effectually  meet  the  situation. 

Succinctly  stated,  the  situation  is  this:  During  the  last 
twenty  years  the  general  death-rate  in  New  York  City  has 
fallen  25  %  and  the  death-rates  from  all  the  principal  causes  of 
deaths  have  fallen  from  10  %  to  35  %,  excepting  in  four  groups 
of  diseases,  in  which  there  has  been  an  increase.  These  are, 
first,  the  acute  respiratory  diseases;  second,  cancer;  third,  the 
diseases  of  the  heart  and  blood-vessels ;  fourth,  the  diseases  of 
the  kidney.  The  increases  in  these  groups  have  been  from  10  % 
to  30  %  or  35  %, — in  the  acute  respiratory  being  from  10  %  to 
15  %.  In  the  first  half  of  1904  there  was  an  extraordinary 
increase  in  this  group,  the  deaths  from  the  acute  respiratory 
diseases  alone  forming  no  less  than  23  %  of  the  total  death-rate. 

The  importance  of  the  situation,  so  far  as  this  group  is  con- 
cerned, is  more  forcibly  brought  out  when  we  compare  the 
percentage  which  the  total  deaths  from  the  acute  respiratory 
diseases  bear  to  the  total  deaths  from  all  causes.     In  1883,  it 


vi  Preface 

was  between  7  and  8  %,  and  in  1893  nearly  16  %,  showing  a 
relative  increase  during  this  period  of  100  %. 

A  consideration  of  the  etiology  of  the  acute  respiratory  diseases 
brings  out  even  more  strongly  the  sanitary  importance  of  the 
problem.  There  can  now  be  no  question  that  the  exciting 
cause  in  each  one  of  the  diseases  of  this  group  is  a  micro-organism, 
which  requires  such  conditions  for  its  growth  and  multiplication 
as  are  usually  found  only  in  the  living  body.  These  organisms 
do  not  under  natural  conditions  multiply  to  any  extent  outside 
of  the  tissues  or  cavities  of  the  body.  The  infection,  when  it 
occurs,  must  practically  always  be  the  result  of  communication, 
directly  or  indirectly,  from  one  human  being  to  another.  The 
conclusion,  therefore,  seems  justifiable  that  these  diseases  are  es- 
sentially communicable,  and  however  great  the  inherent  difficul- 
ties of  the  problem  may  be,  theoretically  at  least,  they  should 
be  to  a  greater  or  less  extent  preventable. 

The  difficidties  in  the  way  of  prevention  arise  largely  from 
the  wide  distribution  of  the  organisms.  Streptococci,  pneumo- 
cocci,  influenza  bacilli,  singly  or  combined,  are  present  in  almost 
all  morbid  conditions  in  the  respiratory  tracts  of  the  inhabitants 
of  large  cities,  and  probably,  also,  to  a  very  large  extent  in  the 
respiratory  tracts  of  healthy  individuals.  The  situation  is  much 
like  that  existing  with  relation  to  diphtheria,  excepting  that 
these  organisms  probably  live  more  readily  and  for  longer 
periods  of  time  in  the  body  cavities  of  healthy  individuals  than 
do  the  diphtheria  bacilli,  and  the  latter  perhaps  have  (at  least  in 
children)  greater  pathogenic  activity. 

The  Board  of  Health,  looking  forward  to  the  study  of  this 
problem  in  the  revision  of  the  Sanitary  Code  made  in  1903,  in- 
cluded this  group  of  diseases  in  the  class  in  which  partly  volun- 
tary and  partly  compulsory  notification  was  required.  This 
provision  of  the  Sanitary  Code,  however,  has  not  yet  been  en- 
forced. It  is  believed  now  that  provision  should  be  made  for 
this. 

It  has  always  been  the  feeling  of  the  Medical  Officers  of  the 
Board,  that  no  really  important  effective  measure  could  be 
taken  in  relation  to  any  preventable  disease  unless  the  Sanitary 


Preface  vii 

authorities  first  had  some  fairly  comprehensive  information  as 
to  the  prevalence  and  distribution  of  the  disease — such  infor- 
mation as  can  onty  be  gathered  from  the  systematic  notification 
of  cases. 

As  the  problem  under  consideration  is  not  simply  one  which 
concerns  New  York,  but  one  which  almost  equally  concerns  all 
the  large  cities  of  the  United  States,  it  was  felt  to  be  important 
that  the  Commission  should  have  a  distinctly  representative 
character,  in  the  hope  that  the  influence  which  it  would  exercise 
would  be  broader  and  more  effective.  As  it  seemed  desirable 
to  the  Commission  to  carry  on  investigations  in  other  labora- 
tories in  other  cities,  suitable  arrangements  were  made  for  de- 
fraying the  expenses  of  these  investigations  by  the  Department 
of  Health  of  New  York  City.  The  facilities  of  the  laboratories  of 
the  Department  of  Health  were  also  placed  at  the  command  of 
the  Commission  for  assisting  in  various  lines  of  investigation  in 
New  York. 

The  Commission  was  constituted  as  follows : 

Dr.  Edward  G.  Janeway,  of  New  York,  President. 

Dr.  William  Osler,  of  Baltimore,  Vice-President. 

Dr.  T.  Mitchell  Prudden,  of  New  York,  Secretary. 

Dr.  Theobald  Smith,  of  Boston. 

Dr.  William  H.  Welch,  of  Baltimore. 

Dr.  Frank  Billings,  of  Chicago. 

Dr.  John  H.  Musser,  of  Philadelphia. 

Dr.  L.  Emmett  Holt,  of  New  York. 

Dr.  Francis  P.  Kinnicutt,  of  New  York. 

(Signed) 

Thomas  Darlington,  M.D., 

President. 

Dr.  Hermann  M.  Biggs, 
General  Medical  Officer, 
Department  of  Health, 
New  York  City. 


INTRODUCTORY  NOTE  TO  STUDIES  ON  THE  PNEUMO- 
COCCUS  UNDER  THE  AUSPICES  OF  THE  MEDICAL 
COMMISSION  FOR  THE  INVESTIGATION  OF  ACUTE 
RESPIRATORY  DISEASES  OF  THE  DEPARTMENT 
OF  HEALTH  OF  THE  CITY  OF  NEW  YORK. 

The  Medical  Commission  for  the  Investigation  of  Acute  Res- 
piratory Diseases,  which  was  formed  at  the  request  of  the  De- 
partment of  Health  of  the  City  of  New  York,  began  its  work  in 
October,  1904. 

It  was  decided  to  concentrate  attention  at  first  upon  lobar 
pneumonia  in  both  its  bacteriological  and  its  clinical  aspects. 
Among  the  studies  upon  the  bacterial  excitant  of  lobar  pneu- 
monia which  it  was  deemed  wise  to  pursue  were  the  following : 

1.  A  study  of  the  occurrence  and  virulence  of  pneumococcus 
and  organisms  related  to  or  resembling  this,  in  the  human 
mouth  in  health  and  disease. 

2.  The  evidence  of  variations  in  virulence  of  the  pneumo- 
coccus. 

3.  The  occurrence  of  the  pnetmiococcus  in  children's  hospitals, 
homes,  and  asylums,  with  a  study  of  the  bacteria  of  mouths 
before  and  after  outbreaks  of  pneumonia. 

4.  The  vitality  of  the  pneumococcus  under  various  conditions. 

5.  A  study  of  mouth  disinfection. 

It  was  the  sense  of  the  Commission  that  one  of  the  earliest 
phases  of  research,  upon  which  much  of  the  subsequent  work 
would  depend,  was  the  determination,  for  the  identification  of 
species,  of  the  characters  of  the  pneumococcus,  this  investiga- 
tion to  include  a  comparative  study  of  the  coccus  from  cases  of 
true  lobar  pneumonia  and  of  that  from  normal  mouths  and 
throats  hitherto  generally  assumed  to  be  identical  with  the 
pneumococcus,  also  to  include  such  a  study  of  streptococci  as 
will  suffice  for  their  separation  from  the  pneumococcus. 


X  Introduction 

The  method  adopted  in  these  studies  was,  first,  to  secure  the 
cooperation  of  bacteriologists  in  various  places,  who  should  make 
independent  studies  along  the  lines  suggested;  and,  second,  to 
establish  a  central  laboratory  or  "clearing  house"  to  which 
ultimately  cultures  from  various  independent  workers  should 
be  sent  for  comparative  study  under  a  single  observer.  It  was 
hoped  that  bv  the  establishment  of  this  central  laboratory  with 
its  large  number  of  available  cultures,  light  might  be  thrown 
upon  atypical  forms,  variations,  etc.,  and  greater  precision 
achieved  in  determining  the  characters  relied  upon  for  identifi- 
cation. 

The  Commission  secured  the  cooperation,  in  making  the  special 
studies  referred  to,  of  Dr.  W.  T.  Longcope,  of  the  Ayer  Clinical 
Laboratory  in  Philadelphia,  of  Dr.  C.  W.  Duval  and  Dr.  Paul 
Lewis  of  the  City  Hospital  in  Boston,  of  Dr.  William  H.  Park 
and  his  associates  in  the  laboratories  of  the  Department  of 
Health  of  New  York  City,  of  Dr.  Leo  Buerger,  of  Mt.  Sinai 
Hospital,  New  York,  of  Prof.  F.  C.  Wood,  of  the  College  of 
Physicians  and  Surgeons,  New  York,  and  Dr.  Charles  Norris,  of 
New  York. 

The  cooperation  of  Prof.  Philip  Hanson  Hiss,  of  the  College 
of  Physicians  and  Surgeons,  New  York,  was  secured  in  the  or- 
ganization and  direction  of  the  work  of  the  central  laboratory  at 
the  College  of  Physicians  and  Surgeons. 

While  various  other  lines  of  study  are  under  way,  it  has  been 
thought  wise  to  publish  at  this  time  together  the  several  in- 
dependent studies  which  follow,  in  the  belief  that  they  may  be 
useful  not  only  in  the  further  work  of  the  Commission  but  of 
others  engaged  in  similar  lines  of  research. 


A  STUDY  OF  PNEUMOCOCCI:  A  COMPARISON  BE- 
TWEEN THE  PNEUMOCOCCI  FOUND  IN  THE 
THROAT  SECRETIONS  OF  HEALTHY  PERSONS  LIV- 
ING IN  BOTH  CITY  AND  COUNTRY  AND  THOSE 
OBTAINED  FROM  PNEUMONIC  EXUDATES  AND 
DISEASED  MUCOUS  MEMBRANES. 

By  WILLIAM  H.  PARK,  M.D.,  Director, 

AND 

A.  W.  WILLIAMS,  M.D.,  Assistant  Director, 

ASSISTED    BY 

A.  OPPENHEIMER,  C.  BOLDUAN,  M.D.,  J.  L.  BERRY,  M.D.,  M.  A.  ASSER- 
SEN,  M.D.,  M.  LOWDEN,  M.D.,  and  I.  VAN  GIESEN,  M.D. 

{From  the  Research  Laboratory  of  the  Health  Department  of  New  York  City.) 

The  investigations  carried  on  in  the  Research  Laboratory  were 
planned  after  consultation  with  the  members  of  the  Commission 
for  the  Investigation  of  Acute  Respiratory  Diseases,  of  the 
Health  Department  of  the  City  of  New  York,  but  were 
otherwise  entirely  independent  of  that  body.  The  study  of  the 
agglutination  characteristics  was  undertaken  by  Dr.  K.  R.  Col- 
hns,  whose  report  follows  this.  The  investigations  are  still  being 
carried  on  and  these  preliminary  reports  are  made  at  the  sug- 
gestion of  the  Commission,  so  that  all  the  workers  in  carrying 
on  further  studies  might  receive  help  from  work  already  done. 

PLAN    OF    INVESTIGATIONS. 

In  this  study  the  following  points  have  been  considered : 

I.  The  presence  of  pneumococci  (i)  in  normal  sputum,  (2)  in 
pneumonic  sputum  and  autopsy  material,  (3)  in  the  sputum  or 
exudates  from  pathogenic  cases  other  than  pneumonias. 

II.  The  comparison  of  the  strains  obtained  from  the  different 
sources  in  the  following  particulars:  (i).  Morphological  and 
cultural  characteristics.     (2).  Virulence.     (3).  Serum  reactions. 

The  scheme  of  the  work,  which  was  carried  out  more  or  less 
fully,  is  tabulated  as  follows : 


TABLE  I. 


^18  i^os  -^  o  -. 

I S 


^^ 

"3 

tx 

3 

c 

-d 

7n 

■d 

3 

/-N 

c 

''^ 

ri 

S 

rt 

o 

> 

1 

(U 

> 

c 

3 

o 

(U 

c 

C 
3 

o 
o 

0 

T 

nto  ca 

and 

-lOOO 

0)               o 
■w      o 

ubcut 

and 

se  (yo 

o 
o 

00 

o 

o 

0 

oc 

o 
6 

6 

o 

o 
d 

+J 

4J 

cu 

s 

n 

Pi 

^ 

^ 

" 

^ 

IH 

<u-M  J3  2  oi  o 
""•d  m  h  t-'  ti 

«  "  o-g  o--; 

g  o  60+^  " 
v:  g  o^-t5  g 

r  "  or;:;       '-' 

+-'  S  ""d  '-' 
V,  o  S  "  ^'^ 
S  h  S  o  o.„ 


t!  S  w  P-.S 
a  o'i3  o  u  o  aj 

*^'d  0)  P  Q,      -M 

"  t-j  d  "     "-"d 


i-i 


^  s 


S  o  2  d  5?  ^  c 


'-;'"T3<umOJ3a)Soc 

Eg.rtrf...SOrt^d-3 
eg      d^      i^i-     •- 

2-^  2  ==  ^-t;  «  S"ti  S^r!  d:5 


■"  c.S 


■^  oi  d 

c.g 


^_^ 

^.^^ 

Jl 

m 

"^ 

1 

bo 

o 

o 

d 

o 

0 

o 

^ 

o 

*^ 

^      o 

d 

IH 

H« 

0 

d 

o 

a 

$ 

^ 

J3 

rt 

C^ 

ts 

g  i 


ts2  K  3  u  o 

(D^  fi  60      m  S 

XI  "^  d    .  d  "* 


(u  iU'i3'd_L 

'3'3  mm  ""^  ''^■d, 
t,  o  <a  *^  u  j3  bo 

'a 


•  S-S^ 


■S"^  is  d  fe  o  "-2 

m  o  O.S  P.^  w  cS 


d  d  m  o  u  o.S  d 
5  o.S      d  d  ts  1) 

ggffid^-^t^C 
(D  P       Q  0_m  (i>  rt 

m^^^u-iTl-^Vl   ^  H 


'•-5;d-o 


d      o  1-  d 
•!=!,;;      o  d  "u-d  S 

s?a  3°  <uw  "*  t- 
l^.ai-d'dgo 

Q  u   CO         f)         r*   ^ 

^^^a^e^^ 

2S^S„§m         . 

w^o.Sddt^.a.s 

ojoS-d-d'SSS 
•^^i5.Sg'-3>1^m 

s 


52 

XI 

d 

oi 

s. 

I-I 

M- 

cu 

<u 

j3 

-d 

X3 

d 

o 

U 

o 

iJ 

(U 

M 

m  m  e  0"m  ^^tS  rtx!  ^-ri  b  C 

s-a|i£^  81^81'^^ 

bo  S  oi  d       m-TSd;  d  O-S  m  ^ 
_g'oy3  d  m  5      •;:  m  oMZi-g  u 

otJ  bo^^S  oJS  <«  o  S'^'o  t«  m 
mdcSooOmd.-5<Uno        '-' 

^  §  a  °  °  a  d|  8  ai  l-^^ 


^g"- 


^     f  >  -*-^    S-iT'     CD     ryT-*^    r 

dyoOCeSot 

drt(nd1Jd3(lJC 


d  M  Mddi-'-So    - 

^ 


'S  C  ^  nJ  u  c!  fl 

l^d'S-l'l 
a^^  a  g-^^- 

^s^sa^i^ 
_s 


so      O  o 
32  .    9 


a  -S^g^ 

"  'aj  C      S-^ 

Sa^^isc^ 


s  „• 


a  "3  rt 


TABLE  II. 

GENEALOGICAL    RECORD. 


O 


Q* 


Q 


._  M  =Inulin-serum  medium  from  ist  rabbit  (description). 


—  M  =  Streak  blood-agar  plate  from  ist  rabbit  (description). 


0 

-(J 

0 

'^ 

Fl 

,0 

^ 

03 
u 

"  ! 

M 

6 

■3322     Inulin-serum    medium    from     3d    rabbit 
(description). 

-2322     Streak   blood-agar   plate   from   3d   rabbit 
(description). 

.1322     Serum-broth  medium  from  3d  rabbit  (de- 
scription). 


'3222     Inulin-serum  medium  from  2d  rabbit  (de- 
scription) . 

-2222     Streak  blood-agar   plate    from    2d  rabbit 
(description). 

-122  2     Serum -broth    medium     from    2d    rabbit 
(description). 


M      Meaning  of  the  numbers  is  the  same  as  the  above,  except 

^  I  instead  of  2  in  units  place  indicates  that  mouse  has 

been   used  instead    of   rabbit,  and]  consequently  the 

numbers  in  hundreds  place,  indicating  the  doses,  have 

•N  a  different  meaning. 


-t->^ 

6 

^ 

0 

rO 

a 

u 

d 

TJ 

0 

<N 

-* 

o 

m  •'-I 
Tt  o 


0 

v 

0 

w 

fl 

^ 

0 

-6 

d 
d 

'0 

„ 

CO 

0 

6 

0 

d" 

0 

;3 

fl 

0 

s 

d 

T) 

d 

<N 

to 

0 

3412 

— 2412 
I4I2 

3312 

2312 

—1312 

■3212 


4  A  Study  of  Pneumococci 

Keeping  of  Records. — Genealogical  tables  of  each  strain  have 
been  kept,  a  modification  of  the  Dewey  Library  System  of  niim- 
bers  being  used  to  indicate  the  cultures.  Thus  by  referring  to 
these  tables  one  is  able  quickly  to  get  the  principal  points  in 
the  history  of  a  particular  culture  from  the  time  of  its  isolation. 

Numbers  of  four  denominations  have  been  used,  the  units 
place  indicating  the  series,  the  tens  place  the  animal  used  for 
inoculations,  the  hundreds  place  the  dose  received,  and  the 
thousands  place  the  medium  employed.  Underneath  this  num- 
ber the  number  of  culture  generations  is  placed  in  parenthesis. 
Table  II  is  one  such  genealogical  table. 

In  addition  to  these  tables,  comment  sheets  on  each  strain 
and  tables  of  comparative  morphology  and  cultural  peculiarities 
have  been  kept. 

THE    PRESENCE    OF    PNEUMOCOCCI. 

Two  hundred  cases  have  been  examined  for  the  presence  of  the 
pneumococcus.  In  the  great  majority  of  cases  two  methods — (a) 
animal  inoculation  of  mass  cultures  and  (6)  stroke  blood-agar 
plates,  as  shown  in  Table  I — ^have  been  employed  in  attempts  at 
isolation ;  in  the  other  cases  only  one  of  these  two  methods  has 
been  used.  Table  III  shows  the  grouping  of  the  cases  and  the  nimi- 
ber  in  which  typical  and  atypical  pneumococci  have  been  foimd. 

From  this  table  we  see  that  typical  pneumococci  have  been 
obtained  in  a  large  percentage  of  normal  cases  in  both  city  and 
country.  A  few  pneumococci  may  have  been  missed  because  of 
occasional  contaminations  or  overgrown  cultures  or  the  employ- 
ment of  large  rabbits  or  some  other  cause.  In  the  majority  of 
cases  where  no  pneimiococci  were  found  streptococci  were  iso- 
lated. From  a  series  of  autopsies  on  cases  of  broncho-pneu- 
monia at  the  Willard  Parker  Hospital,  and  from  a  series  of 
pertussis  sputa  from  the  FoundHng  Hospital,  large  numbers 
of  influenza-like  organisms  were  foimd  with  smaller  numbers  of 
streptococci  and  occasionally  with  a  few  pneumococci.  It  was 
very  difficult  to  get  rid  of  these  influenza-hke  organisms,  as  great 
numbers  passed  through  the  animal  inoculated  with  mass- 
cultures,  and  in  the  plates  {and  >'serum-broth  tubes  they  grew 


William  H.  Park  and  A.  W.  Williams 


abtindantly  in  close  association  with  the  pneumococcus.  Re- 
peated platings  generally  had  to  be  made  before  a  pure  culture 
of  the  pneumococcus  could  be  obtained  in  these  cases. 

TABLE  III. 

SHOWING   NUMBER   OF   CASES    STUDIED   AND   NUMBER   IN   WHICH    PNEUMOCOCCI 

WERE    FOUND. 


Groups. 

Subdivisions. 

Number 

of 

Cases. 

Pneumo- 
cocci  not 
Isolated. 

Pneumococci   Isolated. 

Atypical. 

Typical. 

Normal. 

Research  Laboratory . .  . 

Bellevue  Students 

Saranac  Lake 

3 

lO 

28 

S 

I 

9 
6 

4 
7 
5 
2 

3 
2 

15 
3 

2 
3 

2 

I 
3 

4 
2 

I 

7 
1 1 

Sea  Breeze 

2 

Tarrytown 

I 

Foundling  Hospital    .  .  . 
Briarcliff 

7 
3 

Hyde  Park 

Millbrook 

5 
4 

Newburgh    

Babies'  Hospital 

Pneumonia. 

Lobar- 

53 
21 

4 

5 

5 
2 

45 
14 

Broncho-    

Colds. 

IS 

5 

10 

Miscellaneous . 

Measles 

3 

5 
5 
5 
2 

I 
I 
I 

3 

I 
I 
3 

I 
3 
3 
4 
2 

I 

I 

I 
I 

2 

Scarlet-fever    

2 

Tuberculosis 

2 

Pertussis 

I 

Influenza 

Pleurisy    

I 

Typhoid 

Mastoiditis 

I 

Synovitis    

2 

Meningitis    

I 

CEdema  of  Lungs 

Empyema 

2 

COMPARISON    OF    STRAINS. 

Morphological  and  Cultural  Characteristics. — We  have  divided 
the  pure  cultures  of  pneumococci  obtained  into  two  broad 
groups  according  to  their  morphological  and  cultural  character- 
istics. The  first  group  is  composed  of  typical  pneumococci  and 
the  second  of  atypical  ones. 

By  typical  pneumococci  we  mean  cocci  which  (i)  under  cer- 
tain more  or  less  constant  cultural  conditions  occur  prin- 
cipally in  slightly  elongated  and  pointed  twos  with  broader  ends 


6  A  Study  of  Pneumococci 

apposed,  (2)  under  similar  or  other  cultural  conditions  form 
capsules,  (3)  when  grown  in  inulin-serum  ^  medium  produce  co- 
agulation, and  (4)  when  grown  in  poured  blood-agar  plates  pro- 
duce a  distinct  green  color  in  and  about  colonies. 

By  atypical  pneumococci  we  mean  (i)  cocci  which  morpho- 
logically and  ctdturally  resemble  more  or  less  closely  the  pneu- 
mococcus  except  in  their  growth  in  the  Hiss  inulin  medium, 
which  they  do  not  coagulate;  (2)  cocci  which  are  like  strepto- 
cocci morphologically,  but  which  produce  coagulation  of  the 
Hiss  inulin  medium. 

Referring  to  Table  III  we  see  that  a  larger  number  of  atypical 
strains  have  been  obtained  from  normal  cases,  in  all  of  which 
only  sputum  was  studied,  than  from  pathogenic.  This  may  be 
due  simply  to  the  fact  that  so  many  more  typical  pneumococci 
were  present  in  the  majority  of  pathogenic  cases  studied  that  the 
atypical  ones  may  have  been  missed  in  some  of  these  cases. 
Atypical  pneumococci  of  the  first  group,  i.e.,  those  which  do  not 
coagulate  inulin-serum  medium,  have  been  found  as  the  majority 
of  colonies  and  as  the  only  pneumococcus-like  organism  in  the 
sputum  from  two  cases  of  pneumonia,  and  have  been  accom- 
panied by  typical  pneumococci  in  the  sputum  from  three  other 

'  The  inulin  used  in  the  course  of  the  present  work  in  making  up  Hiss' 
medium  {Jour,  of  Exper.  Med.,  1905,  vii,  317)  was  prepared  in  this  laboratory 
by  R.  B.  Gibson,  for  at  the  time  we  were  unable  to  obtain  it  from  commercial 
sources.  The  method  employed  in  the  obtaining  of  this  substance  follows: 
Dandelion  (taraxacum)  roots  were  soaked  in  cold  tap  water  until  soft,  and  if 
coarsely  ground  the  roots  were  then  run  through  an  ordinary  hash  machine. 
The  material  was  transferred  to  a  gauze  bag,  and  was  washed  thoroughly  in 
running  cold  tap  water  to  remove  a  portion  of  the  soluble  impurities  and  the 
finer  solid  particles  which  would  interfere  with  subsequent  filtration.  The 
washed  roots  were  extracted  in  boiling  water,  strained,  and  filtered.  A  second 
immediate  extraction  followed.  The  filtrates  were  united  and  evaporated  over 
a  Bunsen  burner  to  a  thin  syrup.  Alcohol  (10-15  %)  was  added  and  the  mix- 
ture was  cooled  to  0°  or  below.  The  inulin  separating  out  on  standing  was 
thoroughly  washed  by  decantation  with  cold  alcohol  (10-20  %)  and  then  with 
95  %  alcohol.  It  was  filtered  into  a  suction  funnel,  washed  on  the  filter  with 
hot  alcohol,  sucked  dry,  and  finally  spread  on  filter  paper  in  a  warm  place  to 
remove  the  alcohol  still  remaining.  The  resulting  product  can  be  obtained  as 
a  fine  white  powder  which  gives  the  ordinary  reactions  of  inulin;  solutions  of 
this  product  do  not  reduce  Fehling's  solution.  The  yield  from  five  pounds  of 
the  tubers  was  about  three  hundred  grams. 


William  H.  Park  and  A.  W.  Williams  7 

cases.  So  far  they  have  not  been  found  in  autopsies  following 
pneumonia.  In  one  autopsy  case  and  in  one  broncho-pneu- 
monic sputum  large  numbers  of  cocci  of  the  second  group  of 
atypical  pneumococci  were  found. 

It  is  interesting  to  note  that,  when  some  of  the  non-coagulating 
cultures  were  studied  more  minutely,  various  colonies  being 
fished  and  the  resulting  cultures  being  tested  for  their  ability 
to  coagulate  serum-inulin  media,  in  the  case  of  one  culture  one 
colony  out  of  six  produced  late  coagulation.  From  this  coagulat- 
ing colony,  however,  no  further  colonies  were  obtained  producing 
coagulation.  Among  the  typical  pneumococci  all  strains  vary 
somewhat  with  regard  to  their  power  to  coagulate  the  Hiss 
medium,  a  few  producing  very  late  coagulation.  When  indi- 
vidual colonies  were  fished  from  some  of  these  latter  strains,  it 
was  found  that  there  was  a  wide  variation  in  the  time  required 
for  coagulation,  an  occasional  one  not  coagulating  at  all.  It 
seems,  from  these  observations,  that  the  non-coagulating,  more 
or  less  morphologically  typical  pneumococci  are  closely  related 
to  the  typical  late  coagulators.  One  of  these  atypical  strains 
showed  typical  capsules  in  the  heart's  blood  of  animals  and  the 
others  showed  occasional  small  capsules. 

All  of  the  typical  and  atypical  strains,  as  well  as  many  strains 
of  streptococci,  have  produced  a  green  color  in  and  about  colo- 
nies in  poured  blood-agar  plates,  while  other  streptococci  have 
produced  large  areas  of  haemolysis  about  colonies  and  no  green 
color.  These  results  agree  in  part  with  those  of  Schottmiiller 
(Munchener  med.  Woch.,  1903,  p.  849),  and  E.  Frankel 
{Milnchener  med.  Woch.,  1905,  p.  548),  who  divide  strepto- 
cocci into  three  groups  according  to  their  behavior  in  blood-agar 
plates:  Streptococcus  pyogenes  producing  much  haemolysis. 
Streptococcus  virideus  producing  green  color,  and  Streptococcus 
mucosus  producing  mucous-like  material  as  well  as  green  color. 
These  results  differ  from  those  of  Rosenow  {Journal  of  Infec- 
tious Diseases,  1904,  I,  280)  who  states  that  no  streptococcus 
tried  by  him  produced  green  color,  while  all  pneumococci  did, 
and  he  therefore  recommends  this  test  in  differentiating  the 
two  species.     From  the  sputa  of  a  number  of  cases  of  broncho- 


8  A  Study  of  Pneumococci 

pneumonia  we  tried  to  isolate  the  pnetimococcus  by  this  method, 
making  poured  blood-agar  plates  from  different  dilutions  of  the 
sputum  and  fishing  from  the  green  colonies,  and  at  the  same  time 
we  used  the  method  of  animal  inoculation  by  mass-cultures. 
In  every  case  by  the  first  method  only  streptococcus-like  organ- 
isms were  obtained,  while  by  the  second  typical  pneumococci 
were  isolaLed. 

All  of  the  strains  of  typical  pneumococci  studied  by  us  may 
be  divided  into  several  distinct  morphologic  varieties.  We  call 
them  varieties,  because  while  each  strain  shows  a  wide  limit  of 
fluctuating  variability,  certain  strains  have  similar  predomi- 
nating constant  characteristics.     These  varieties  are: 

1.  Small  cocci  occurring  under  most  cultural  conditions  in 
twos  and  producing  small  capsules. 

2.  Large  cocci  occurring  readily  in  short  and  medium-length 
chains  and  producing  large  capsules. 

3.  The  so-called  Streptococcus  mucosus. 

The  first  two  varieties  are  less  distinctly  bounded  than  the 
last  which  forms  a  definite  morphologic  variety.  This  variety, 
which  has  been  mentioned  only  a  few  times  in  literature  (Schott- 
miiller,  Miinchener  med.  Woch.,  1903;  L.  Buerger,  Medical  News, 
1904,  p.  1117;  E.  Frankel,  Miinchener  med.  Woch.,  No.  12,  1905; 
L.  Heim,  Zeit.  fiir  Hyg.,  1905,  I,  139),  has  been  classed  as  a 
streptococcus,  under  the  name  of  Streptococcus  mucosus  by 
Schottmiiller,  and  Streptococcus  mucosus  capsulatus  by  others. 
It  has  been  isolated  by  us  from  eight  cases  of  pneumonia,  from 
two  cases  of  cold,  and  from  two  normal  individuals,  and  has 
been  seen  in  mixed  cultures  in  a  number  of  other  cases.  Three 
of  the  cases  of  pneumonia  were  early  autopsy  cases.  In  two 
of  these  the  organism  occurred  pure  and  in  large  numbers  (two 
hundred  colonies  were  fished  in  one  case  and  the  resulting 
cultures  were  all  similar) ;  in  the  third  case  it  was  accompanied 
by  a  smaller  number  of  the  first  variety  of  t^^pical  pnuemococci. 
In  one  pneumonic  sputum  and  in  one  normal  individual  the  first 
variety  of  typical  pneumococci  also  accompanied  it,  while  in  all 
the  other  cases  it  was  the  only  pneumococcus-like  organism 
isolated.     It  has  thus  been  found  by  us  more  frequently  in 


William  H.  Park  and  A,  W.   Williams  9 

cases  of  pneumonia  than  in  other  cases.     We  have  classed  it 
among  the  typical  pneumococci  for  the  following  reasons : 

1.  On  serum-free  culture  media  after  the  first  two  or  three 
culture  generations  it  produces  no  mucous-like  material  and 
shows  no  capsule  or  chain  formation,  but  appears  like  a  typical 
pneumococcus. 

2.  It  readily  coagulates  the  Hiss  inulin  medium. 

3.  It  shows  very  distinct  capsules  in  serum  media  and  in  the 
blood  of  inoculated  animals. 

4.  It  has  been  found  pure  and  in  large  numbers  in  two  cases 
of  typical  lobar  pneumonia. 

5.  The  results  obtained  from  absorption  experiments  (see  the 
Collins  report)  indicate  a  close  relationship  between  it  and  certain 
typical  pneumococci  of  the  second  variety,  while  no  relationship 
is  shown  between  it  and  the  strains  of  typical  streptococci  tested. 

It  has  been  classed  with  the  streptococci  heretofore  because 
of  its  ability  readily  to  produce  rounded  forms  and  short  chains. 
According  to  our  descriptions  of  typical  and  atypical  pneumo- 
cocci it  might  be  classed  by  many  with  the  latter,  but  considering 
its  ability  under  certain  conditions  to  show  typical  pneumococcus 
forms  we  prefer  to  class  it  with  the  former,  and  make  it  a  distinct 
variety.  With  regard  to  nomenclature,  it  should  be  called, 
according  to  the  classification  followed.  Streptococcus  lanceo- 
latus,  var.  mucosus  (the  classification  of  Lehmann  and  Neu- 
mann, which  we  prefer),  or  Diplococcus  lanceolatus,  var.  mucosus 
(the  classification  of  other  authors) ;  we  have  given  it  the  trivial 
name,  Pneumococcus  mucosus. 

By  refering  to  the  section  on  serum  reactions  below  and  to  Dr. 
Collins 's  report  on  the  agglutination  of  the  pneumococcus,  it  will 
be  found  that  all  the  strains  of  this  variety  isolated  by  us  show 
a  specific  similarity  in  these  reactions. 

A  certain  number  of  cultures  from  both  normal  and  abnormal 
cases,  which  showed  the  characteristics  of  typical  pneumococci 
immediately  after  isolation,  have  later  dropped  some  of  these 
characteristics  and  become  more  like  streptococci.  They  appear 
principally  in  chains  and  no  longer  coagulate  the  inulin-serum 
medium.     Whether  some  of  these  cultures  were  mixed  in  the 


10  A  Study  of  Pneumococci 

beginning  with  a  streptococcus-like  organism  growing  in  inti- 
mate connection  with  the  pneumococcus,  as  the  influenza  baciUus 
does,  and  finally  outgrowing  it,  or  whether  they  are  all  mutating 
varieties,  is  still  a  question.  With  such  a  mass  of  cultures  it  was 
impossible  to  follow  each  closely,  to  make  plates,  and  to  study 
colonies  of  each  new  culture  generation,  but,  judging  from  the  few 
apparently  changing  strains  which  have  been  more  minutely 
studied,  it  would  seem  as  if  some  of  these  cultures  were  really 
changing  by  mutation.  None  of  them  have  become  perma- 
nently typical  streptococci — that  is,  they  show  more  or  less 
irregularity  in  chain  production,  sometimes  produce  elongated  and 
pointed  twos  and  always  green  color  in  blood-agar  plates,  but  they 
seem  gradually  to  lose  their  power  to  coagulate  inulin-serum 
medium.  These  obser\^ations  in  regard  to  mutating  varieties  in- 
dicate a  close  relationship  between  certain  pneimiococci  and  strep- 
tococci, a  relationship  which  previous  investigators  have  noted. 

All  strains  of  pneumococci  tried  coagulated,  usually  within 
forty-eight  hours,  serum  media  containing  dextrose,  lactose,  or 
saccharose,  as  do  also  certain  strains  of  streptococci.  With 
mannit  different  strains  of  pneumococci  act  differently.  Out 
of  one  hundred  strains  tested,  tw^enty-nine  did  not  coagulate 
mannit-serum  mediimi  after  fourteen  days.  Among  the  seventy- 
one  coagulators,  sixteen  coagulated  in  tw^enty-four  hours,  seven- 
teen in  forty-eight  hours,  one  on  the  third  day,  five  on  the  fifth 
day,  and  the  rest  betAveen  the  fifth  and  fourteenth  days.  With  the 
exception  of  the  mucosus  variety,  there  seems  to  be  no  relation  be- 
tween this  coagulation  and  the  varieties  or  groups  of  pneumococci. 
All  of  the  Pneumococcus  mucosus  strains  tested  coagulated  the 
mannit  mediimi  within  two  days.  Certain  atypical  strains  which 
did  not  coagulate  the  inulin  readily  coagulated  the  mannitmedium, 
while  the  few  definite  streptococci  tried  did  not  coagulate  either. 
The  plate  growths  from  these  non-coagulating  cultures  all  showed 
practically  as  many  colonies  as  those  from  the  coagulating  ones. 

Virulence. — The  virulence  of  the  different  strains  of  pneumo- 
cocci for  lower  animals  depends  in  great  measure  upon  the 
method  of  isolation  used.  If  the  plate  method  be  employed, 
fishing  individual  colonies,  the  majority  of  pure  cultures  ob- 


William  H.  Park  and  A.  W.  Williams  11 

tained  will  be  distinctly  less  virulent  than  those  isolated  by  the 
mass-culture  method.  The  mass-culture  method  consists  in 
inoculating  a  mass  of  sputum  or  material  to  be  tested  into 
serum-broth  (previously  tested  for  ability  to  give  abundant 
growth  of  pneumococci) ,  placing  at  36°  C.  for  twenty-four  hours, 
and  inoculating  a  certain  amount  of  the  resulting  culture  sub- 
cutaneously  into  the  animal  chosen.  The  culture  isolated  from 
the  heart's  blood  of  the  animal  at  autopsy  is  then  tested  for 
virulence  in  the  same  species  of  animal. 

We  have  used  both  rabbits  and  white  mice  for  the  inoculations, 
but  in  the  great  majority  of  cases  the  former  animals  only. 
Young  rabbits,  weighing  from  800  to  1000  grams,  and  young 
adult  mice  have  been  chosen. 

By  testing  the  virulence  of  strains  isolated  by  the  mass-culture 
method,  it  has  been  shown  that  the  percentage  of  virulent  strains 
of  pneumococci  isolated  from  cases  of  pneumonia  is  higher  than 
the  percentage  of  those  isolated  from  normal  cases  (see  Table  IV) . 


TABLE  IV. 

PERCENTAGE    OF    VIRULENT 

STRAINS. 

Amount  Inoculated. 

Pneumonia  Cases. 

Healthy  Individuals, 

4.0  C.C. 

O.I  c.c. 

87% 
51% 

69% 
31% 

Most  of  the  strains  isolated  from  the  cases  of  broncho-pneu- 
monia which  are  included  with  the  cases  of  pneumonia  are  not 
very  virulent,  while  most  of  the  strains  from  the  colds  which 
have  not  been  noted  here  are  virulent.  Among  the  normal 
individuals  the  largest  percentage  of  virulent  pneumococci  came 
from  the  Foundling  Hospital  children,  the  next  from  the  Bellevue 
students,  the  next  from  the  country  around  New  York,  and  the 
smallest  from  Saranac  Lake.  Too  much  weight  should  not  be 
attached  to  this  summary,  because  of  the  comparatively  small 
number  of  cases  examined. 

Normal  No.  40  in  contact  with  Pneumonia  No.  36  were  of 
equally  extreme  virulence  for  both  mice  and  rabbits.     All  of  the 


12  A  Study  of  PneuTYiococci 

Pnetunococcus  mucosus  strains  tested  have  been  with  one  excep- 
tion extremely  virulent  for  mice  and  decidedly  less  so  for  rabbits. 

Retention  of  Virulence. — Grown  on  artificial  media,  all  of  the 
virulent  strains  are  losing  their  virulence  although  those  trans- 
planted on  media  containing  blood  from  the  species  of  animal  used 
for  the  testing  remained  more  virulent  longer,  for  that  species  than 
for  the  other  species  of  test  animal  chosen.  No.  36,  however,  one 
of  the  most  virulent  strains  tested,  remained  virulent  for  a  long 
time  for  both  rabbits  and  mice  when  grown  on  rabbit  blood-agar. 
It  seems  now  gradually  to  be  losing  its  virulence  for  both  animals. 
When  grown  in  Bolduan's  calcium-broth  medium  {New  York 
Med.  Jour.,  May,  1905),  cultures  of  pnetunococcus  remain  alive 
and  retain  their  virulence  as  long  as  when  grown  in  senun-broth 
according  to  the  few  tests  made;  therefore,  as  this  medium 
generally  allows  an  abundant  growth,  it  is  an  excellent  one  to  use 
when  for  any  reason  the  use  of  serum  is  undesirable. 

Agglutination  reactions  are  described  in  a  separate  report  by 
Dr.  Collins. 

SERUM    REACTIONS. 

Specific  Protective  Substances. — According  to  Neufeld  and 
Rimpau  {Deutsche  med.  Woch.,  1904,  p.  1458),  the  senun 
of  rabbits  inoculated  with  pneumococcus  cultures  becomes 
speedily  protective  for  white  mice.  They  claim  to  have 
obtained  after  the  second  inoculation  of  large  doses  of  pneu- 
mococcus bodies,  the  first  killed  by  heat  and  the  second  living,  a 
serum  which  was  highly  protective  for  mice.  They  claim  that 
this  serum  has  no  lytic  properties  for  the  pneumococcus,  but 
that  the  specific  protective  substance  is  a  bacteriotropic  sub- 
stance uniting  with  the  bacteria  and  preparing  them  for  ingestion 
by  the  leucocytes,  and  that  when  this  serum  is  added  to  a  mixture 
of  bacteria  and  normal  leucocytes  in  vitro  more  phagocytosis  is 
produced  than  when  normal  serum  is  used.  So  far  Neufeld's  bac- 
teriotropic substance  agrees  with  Wright's  {Proceedings  of  the  Royal 
Soc.  of  London,  1903,  LXXII,  337)  opsonic  substance,  except  that 
Neiifeld  claims  that  his  substance  is  "not  destroyedby  low  heat,  while 
Wright  says  that  his  is.  Therefore  Neufeld  states  that  his  bacterio- 
tropic substance  is  not  the  same  as  Wright's  opsonic  substance. 


William  H.  Park  and  A.  W.  Williams  13 

Very  little  as  A^et  has  been  done  by  us  in  attempting  to  raise 
in  animals  specific  protective  substances  for  the  pneumococcus 
or  in  studying  the  properties  of  such  substances.  In  the  begin- 
ning we  followed  Neufeld's  method,  inoculating  large  doses  of 
surface  cultures  of  pnetmiococci  into  rabbits.  The  first  cultures 
were  subjected  to  from  60°  to  65°  C.  for  from  fifteen  to  thirty 
minutes  and  the  subsequent  cultures  were  hving.  There  is  no 
doubt  that  a  preliminary  large  dose  of  a  dead  culture  could  be 
followed  by  a  larger  dose  of  a  living  culture  without  causing 
death  than  if  a  small  preliminary  dose  had  been  used,  but  the 
serum  of  such  rabbits  showed  no  protective  action  in  mice  with 
any  of  the  strains  of  pneumococcus  tested.  Only  a  few  tests 
were  made,  however,  so  no  definite  conclusion  can  be  drawn. 
The  phagocytic  power  in  vitro  seemed  to  be  slightly  increased 
for  some  of  the  strains,  each  by  its  own  serum. 

It  was  found  that  the  opsonic  power  of  normal  rabbit,  sheep, 
and  especially  of  normal  horse  serum  is  very  great  for  some 
strains  of  pneimiococci,  less  so  for  others,  and  very  slight  for 
others.  All  of  the  strains  of  Pneumococcus  mucosus  tested  belong 
to  this  last  group.  Since  rabbits  proved  unsatisfactory,  it  was 
decided  to  experiment  with  sheep.  Two  sheep  were  chosen,  one 
of  which  was  inoculated  with  one  of  the  first  variety  of  pneumo- 
coccus and  the  other  with  a  strain  of  Pneumococcus  mucosus. 
The  sheep  have  received  eleven  inoculations  and  have  been  bled 
twelve  times.     (See  Table  V.) 

The  serum  from  each  bleeding  was  tested  in  vitro  for  its  opsonic 
or  bacteriotropic  power  on  a  number  of  strains  of  pneumococci, 
and  from  a  few  of  the  bleedings  it  was  tested  in  addition  for  its 
protective  pov/er  in  white  mice.  In  testing  the  opsonic  power 
of  the  serum  in  vitro  the  following  technic  was  used:  To  0.5  c.c 
of  serum,  undiluted  or  diluted,  in  a  short  wide  test  tube,  were 
added  0.5  c.c.  of  a  thick  suspension  of  washed  normal  leucocytes 
and  0.5  c.c.  of  the  dilution  of  bacteria.  The  washings  and  dilu- 
tions were  made  with  0.8  %  of  sodium  chloride  solution.  The 
mixtures  were  kept  at  36°  C.  and  smears  made  at  stated  times. 
The  leucocytes  almost  immediately  form  a  thin  layer  about 
the  sides  and  bottom  of  the  test-tube  and  a  well  spread  smear 


14 


A  Study  of  Pneumococci 


containing  large  numbers  of  leucocytes  is  made  by  scraping  from 
this  layer  with  a  flatty  coiled  platinum  loop  and  spreading 
quickly  on  a  clean  glass  slide.  The  smears  were  fixed  in  methyl 
alcohol  and  stained  with  eosin  and  methylene  blue.  Normal 
leucocytes  from  rabbits,  guinea-pigs,  sheep,  and  horses  have  been 
used,  and  so  far  our  results  have  agreed  with  those  of  all  other 

TABLE  V. 

INOCULATIONS    AND    BLEEDINGS    OF    SHEEP. 


Amount  Inoculated. 


20  c.c.  of  24-hr.  broth  cult,  centrifugalized  and  exposed 
to  60°  C.  for  20  min 

27  c.c.  of  24-hr.  broth  cult,  centrifugalized  and  exposed 

to  60°  C.  for  20  min _. 

28  c.c.  of  24-hr.  broth  cult,  centrifugalized  and  exposed 

to  60°  C.  for  10  min 

32  c.c.  of  24-hr.  calcium-broth  cult,  centrifugalized.  .  .  . 

40  c.c.  of  24-hr.  calcium-broth  cult.,  30  c.c.  centrifu- 
galized and  10  c.c.  non-centrifugalized 

50  c.c.  of  24-hr.  calcium-broth  cult.,  30  c.c.  centrifu- 
galized and  20  c.c.  non-centrifugalized 

60  c.c.  of  24-hr.  calcium-broth  cult.,  30  c.c.  centrifu- 
galized and  30  c.c.  non-centrifugalized 

70  c.c.  of  24-hr.  calcium-broth  cult.,  30  c.c.  centrifu- 
galized and  40  c.c.  non-centrifugalized 

80  c.c.  of  24-hr.  glucose-calcium-broth  cult.,  30  c.c. 
centrifugalized  and  50  c.c.  non-centrifugalized. . .  . 

85  c.c.  of  24-hr.  glucose-calcium-broth  cult.,  30  c.c. 
centrifugalized  and  55  c.c.  non-centrifugalized. . .  . 

50  c.c.  of  24-hr.  glucose-calcium-broth  cult.,  -Hio  slant 
blood-agar  cult 


Date  of 
Inoculation. 

Date  of 
Bleeding. 

March    3 



March  10 

March  15 

17 
"        29 

24 
April      2 

April      8 

— 

19 

April    2  7 

"        28 

May       4 

May    5 

"       10 

"        15 

24 

"       27 

June       s 

June      8 

"       15 

— 

"       21 

observers  in  regard  to  the  indifferent  action  of  leucocytes  from 
different  species  of  animals.  According  to  our  experiments, 
some  species  of  leucocytes  need  more  careful  washing  than  others, 
probably  because  of  the  greater  opsonic  power  of  the  correspond- 
ing normal  serum.  For  example,  horse  leucocytes  must  be  most 
carefully  washed  in  order  to  keep  the  controls  from  showing 
phagocytosis.  We  have  used  horse  leucocytes  for  many  of  the 
experiments  because  of  our  ability  to  obtain  them  easily  and 
quickly  in  large  quantities.  The  horse  is  bled  just  before  the 
leucocytes  are  to  be  used  and  the  blood  is  collected  aseptically  in 
flasks,  with  one  tenth  its  volume  of  a  lo  %  solution  of  sodium 


William  H.  Park  and  A.  W.  Williams  15 

citrate  in  normal  salt  solution.  After  mixing,  the  blood  is  allowed 
to  stand,  and  within  ten  minutes  the  red  blood  cells  have  settled, 
leaving  the  plasma,  containing  many  leucoc3rtes,  above.  This  is 
drawn  off,  centrifugalized,  and  the  leucocytes  are  washed  care- 
fully four  times;  each  time  fresh  sterile  plugs  are  used  for  the 
tubes.  In  this  way  it  is  easy  to  obtain  a  large  amount  of  a 
very  thick  suspension  of  actively  motile  polynuclear  leucocytes. 
Of  such  a  suspension  0.5  c.c.  added  to  the  mixture  of  0.5  c.c. 
each  of  0.8  %  salt  solution  and  the  required  dilution  of  bacteria 
has  been  used  as  one  control,  and  a  similar  mixture  with  normal 
serum  in  the  place  of  the  0.8  %  salt  solution  as  another. 

The  dilutions  of  the  bacteria  were  prepared  as  follows:  A 
twenty-four-hour  calcium-broth  culture  made  from  a  twenty- 
four-hour  blood  agar-slant  culture  of  the  stock  culture  (the  blood- 
agar  was  made  from  rabbit  blood  and  kept  in  the  thermostat  at 
36°  C.  for  two  days  before  using)  was  centrifugalized  and  enough 
0.8  %  salt  solution  added  to  the  bacteria  to  make  a  suspension  of 
about  2,000,000,000  bacteria  to  the  cubic  centimeter. 

In  estimating  the  phagocytic  action  by  this  method,  it  has 
been  found  that  a  large  number  of  polynuclear  leucocytes  must 
be  counted,  as  phagocytosis  seems  to  occur  irregularly,  a  group 
of  polynuclear  leucocytes  each  one  loaded  with  bacteria  filling  one 
field,  and  a  group  containing  no  organisms  the  next. 

The  mixtures  were  examined  in  the  beginning,  at  the  end  of 
one  quarter,  one  half,  two,  three,  five,  and  twenty-four  hours. 
It  was  found  that  the  difference  between  the  serum  controls  or 
heated  serum  and  the  specific  serum  was  more  marked  after 
fifteen  to  thirty  minutes  than  at  the  height  of  phagocytosis, 
which  occurred  in  from  two  to  three  hours.  At  the  latter  time 
the  differences,  if  any,  were  very  slight.  The  specific  serum 
thus  seemed  to  allow  the  phagocytosis  to  occur  more  quickly. 

The  difference  between  the  opsonic  power  in  vitro  of  the  normal 
serum  and  the  specific  serum,  however,  has  so  far  been  slight. 
This  slight  increase  of  opsonic  power  of  the  specific  serum  was 
apparent  after  the  second  bleeding  and  continued  up  to  and  in- 
cluding the  eighth  bleeding,  but  the  serum  from  the  next  two 
bleedings   (ninth  and  tenth)    showed   no  definite  difference  in 


16  A  Study  of  Pneumococci 

phagocytosis  between  normal  serum  controls  and  specific  serum. 
The  serum  from  the  ninth  bleeding,  however,  showed  a  protective 
power  for  mice  similar  to  that  of  the  serum  from  the  eighth 
bleeding,  and  the  serum  from  the  tenth  bleeding  prolonged  life. 
As  the  control  animals  in  these  experiments  all  died,  the  absolute 
protective  power  of  these  sera  is  not  known.  From  these  data 
all  that  can  be  said  is  that  while  the  phagocytic  power  in  vitro  of 
a  certain  specific  serum  seemed  no  greater  than  that  of  the 
control  serum,  yet  the  former  possessed  marked  protective 
power  in  mice.  One  of  the  heterologous  strains  (Pn.  4)  showed 
clumping  and  marked  phagocytosis  with  Sheep  Serum  II  (inocu- 
lated with  Pneumococcus  mucosus),  while  with  Sheep  Serum  I 
(inoculated  with  Pn.  36)  it  showed  no  clumping  and  less  phagocy- 
tosis, and  yet  mice  were  protected  from  it  by  this  latter  serum. 
All  of  the  Pneumococcus  mucosus  strains  showed  very  slight 
phagocytosis  in  any  serum,  and  yet  with  Sheep  Serum  II  mice 
were  protected  from  all  of  the  strains  with  but  one  exception. 

It  seems  from  these  observations  that  the  degree  of  phagocy- 
tosis in  vitro  with  some  sera  at  least  is  not  an  indication  of  the 
degree  of  protective  power  in  mice. 

In  regard  to  the  influence  of  heat  upon  the  phagocytic  power 
of  these  sera,  the  following  results  have  been  obtained:  60°  C. 
for  a  half  hour  has  slight  deleterious  effect,  65°  for  twenty  min- 
utes has  more,  and  60°  for  one  hour  has  a  marked  effect. 

Poured  blood-agar  plates  after  two  hours  at  36°  C.  show  a 
decrease  in  the  niunber  of  colonies  with  all  the  strains  which 
agglutinated,  but  the  decrease  is  no  greater  than  could  probably 
be  accounted  for  by  the  agglutination. 

SUMMARY    AND    CONCLUSIONS. 

1.  Typical  pneumococci  were  present  during  the  winter 
months  in  the  throat  secretions  of  a  large  percentage  of  healthy 
individuals  in  city  and  country. 

2 .  A  higher  percentage  of  atypical  strains  of  pneumococci  have 
been  obtained  from  healthy  persons  than  from  those  suffering 
from  pneumonia.  In  the  latter  cases  the  atypical  strains  may 
have  been  overlooked,  because  of  the  larger  number  of  typical 


William  H.  Park  and  A.  W.  Williams  17 

pneumococci  present.     Many  of   the   atypical  strains  seem  to 
be  closely  related  to  the  streptococci. 

3.  The  so-called  Streptococcus  mucosas  Schottmiiller,  which 
has  hitherto  been  classed  with  the  distinct  streptococci,  is  placed 
as  a  definite  variety  among  the  pneumococci,  and  it  is  recom- 
mended that  the  name  be  changed  to  Streptococcus  lanceolatus, 
var.  mucosus. 

4.  A  lower  percentage  of  strains  of  pneumococci  virulent  for 
rabbits  in  the  doses  used  has  been  obtained  from  normal  cases 
by  rabbit  inoculations  of  mass  cultures  than  from  cases  of  pneu- 
monia by  the  same  method. 

5.  Since  the  virulence  of  pneumococci  may  be  rapidly  in- 
creased for  a  susceptible  species  of  experimental  animal  by  suc- 
cessive passage,  and  since  pneumococci  obtained  from  most 
pneumonias  are  more  virulent  for  experimental  animals  than 
are  those  obtained  from  healthy  individuals,  therefore  the  viru- 
lence of  pneumococci  from  cases  of  human  infection  is  probably 
increased  for  human  beings ;  hence  cases  of  pneumonia  should 
be  considered  to  a  certain  degree  as  contagious  and,  since  the 
virulence  of  the  pneumococcus  may  be  quickly  increased  and 
since  the  organism  is  very  prevalent  in  normal  sputum,  all  pos- 
sible measures  should  be  taken  to  restrict  public  expectoration. 

6.  By  repeated  inoculations  into  sheep  of  a  pneumococcus 
strain,  a  specific  protective  power  of  this  serum  for  mice  is  devel- 
oped against  the  homologous  strain  and  against  certain  other 
strains,  one  morphological  variety  (Streptococcus  lanceolatus,  var. 
mucosus)  being  thus  clearly  differentiated  from  other  strains. 

7 .  Coincident  with  this  production  of  protective  power,  a  slight 
specific  increase  of  the  sheep  serum  in  phagocytic  power  in  vitro 
has  been  observed  with  some  strains  of  pneumococci,  all  strains 
of  Streptococcus  lanceolatus,  var.  mucosus,  acting  similarly  with 
the  serum  produced  by  the  inoculation  of  one  strain ;  the  strains 
of  some  other  varieties,  however,  have  shown  no  definite  rela- 
tionship between  the  phagocytic  power  and  the  protective 
power  of  the  serum. 

Note. — The    protocols  of  cases  and  experiments  will  be  published  later  in 
the  Reports  of  the  Health  Department  of  New  York  City. 


THE  APPLICATION   OF  THE   REACTION  OF  AGGLU- 
TINATION TO  THE  PNEUMOCOCCUS. 

'-  By  KATHERINE  R.  COLLINS,  M.D., 

Assistant  Bacteriologist. 

{From  the  Research  Laboratory  of  the  Department  of  Health,  New  York  City.) 

The  following  report  is  a  part  of  the  work  on  pneumonia  as 
planned  by  the  Commission  for  the  Investigation  of  Acute  Respi- 
ratory Diseases,  and  by  the  Department  of  Health. 

Normal  serum  of  various  animals  differs  greatly  in  its  tendency 
to  agglutinate  many  strains  of  pneumococci.  Thus  rabbit  serum 
generally  gave  negative  results,  while  sheep  and  horse  serum 
reacted  slightly  in  a  few  instances,  and  the  serum  of  one  goat  of 
fotir  tested  agglutinated  a  number  of  strains  of  pneumococci  in 
dilutions  of  i :  lo. 

Of  two  normal  himian  sera  tested,  one  failed  to  react  while 
the  other  agglutinated  several  of  the  organisms  in  dilutions  as 
high  as  I :  lo. 

Neufeld,  Clairmont,  Landsteiner,  Wadsworth,  Heyrovsky, 
and  others  have  succeeded  in  producing  agglutinins  for  pneumo- 
cocci in  the  animal  body  through  immunization. 

Gorgano  and  Fattori  state  that  agglutination  of  Diplococcus 
pneumoniae  with  the  blood  of  patients  suffering  from  infection 
with  this  organism  is  constant,  that  it  persists  for  some  time 
after  recovery  of  the  patient,  and  that  the  reaction  is  more 
marked  if  the  homologous  organism  be  used.  The  highest 
reaction  obtained  by  them  was,  however,  in  a  i :  lo  dilution. 

Glucose  broth  has  been  generally  recommended  as  the  medium 
best  adapted  for  making  agglutination  tests  with  the  pneumo- 
coccus,  but  as  the  organism  quickly  dies  out  in  the  presence  of 
the  excess  of  acid  produced  by  the  fermentation  of  the  sugar, 
it  can  be  carried  through  one  generation  only  on  this  medium, 

i8 


Katlierine  R.  Collins  19 

iinless  it  is  transferred  before  the  acidity  has  increased  suffi- 
ciently  to   destroy   the   growth.     This   fact   makes   the   broth, 
unsuitable  for  the  work. 

Marshall  and  Knox  ^  and  Morello  have  shown  that  the  typhoid 
bacillus  loses  its  agglutinability  when  grown  for  some  time  in  an 
active  immtme  serum.  Dr.  Park  and  I  have  demonstrated  the 
same  to  be  true  for  the  bacillus  of  dysentery  when  grown  in  its 
immune  serum,  and  we  were  also  able  to  show,  further,  that 
the  agglutinability  could  be  restored  by  long  cultivation  upon 
suitable  media. 

As  the  presence  of  serum  constituents  in  the  medium  is  re- 
quired for  the  continuous  growth  of  the  pneumococcus,  it  might 
be  assumed  from  the  above  facts  that  the  agglutinability  of  the 
organism  might  at  least  be  lowered  by  long  cultivation  upon  a 
medium  containing  even  very  small  amounts  of  these  inhibitory 
substances.  This  assumption  was  borne  out  by  several  tests 
made  with  an  organism  taken  on  the  one  hand  directly  from  a 
fresh  rabbit-blood-agar  culture,  and  on  the  other  from  a  culture 
in  calcium  broth  2  one  generation  old  and  several  generations  old. 
The  last  culture  gave  the  best  reaction,  while  the  culture  from 
the  blood-agar  gave  the  poorest  reaction. 

To  eliminate  this  source  of  error,  diluted  sheep  or  hog  serum, 
as  suggested  by  Dr.  Park,  was  boiled  to  destroy  any  inhibitory 
substances  present,  and  added  to  broth  or  agar  as  the  case  re- 
quired. Cultures  obtained  from  media  containing  these  sera, 
when  transferred  to  calcium  broth,  usually  gave  a  homogeneous 
growth  with  much  less  tendency  to  spontaneous  agglutination 
than  is  seen  with  cultures  grown  in  calcium-glucose  broth. 
Heating  the  organism  to  70°  C.  for  15  minutes  does  not  affect 
its  agglutinability.     Heating  the  serum   to  85°  for  15  minutes, 

1  The  studies  of  Marshall  and  Knox,  so  far  as  I  know,  have  not  as  yet  been 
published. 

2  The  fact  that  the  addition  of  calcium  carbonate  to  culture  media  neutralizes 
the  acid  formed  by  the  fermentation  of  sugar  dvuing  bacterial  growth  has  been 
recognized  for  some  time.  The  application  of  this  reaction  to  the  growth  of 
the  pneumococcus  was  suggested  independently  by  Bolduan  and  Hiss.  The 
former  recommended  the  addition  of  bits  of  marble  to  plain  broth,  the  latter 
used  calcium  carbonate  in  the  forra  of  powder  in  glucose  broth. 


20 


Reaction  of  Agglutination  to  the  Pneumococcus 


however,  destroys  the  agglutinins  both  for  the  pneumococcus  and 
the  Pneumococcus  mucosus.^ 

Several  methods  of  immunization  were  tried.     The  one  which 
gave  the  best  results  is  represented  by  the  following  example : 

Feb. 


15- 


Feb. 

2<?. 

Marc? 

,    8 

" 

13 

" 

24 

April 

I 

" 

8 

" 

15 

" 

25 

May 

I 

" 

8 

" 

15 

" 

21 

May     22 


A  rabbit  was  given  subcutaneously  3   c.c.  of  a  culture  grown  for 
48  hours  in  broth  to  which  a  few  drops  of  defibrinated  blood 
were  added,  heated  previously  to  60°  C.  for  30  minutes. 
5  c.c.  of  a  similar  culture  administered. 
\  c.c.  of  a  living  culture  of  the  same  organism  was  given. 
I  c.c.     "         "  "         "     "        "  "  "         " 

5  c.c.  of  a  calcium-broth  culture  heated  70°  C.  for  15  minutes  given. 
5  c.c. 
5  c.c. 
10  c.c. 
10  c.c. 
10  c.c. 
IS  c.c. 
15  c.c. 
Animal  bled  and  the  serum  tested  with  the  homologous  organism 

which  it  agglutinated  in  a  dilution  of  i:  200. 
An  emulsion  heated  to  70°  C.  for   15  minutes  from  four  heated 
serum-agar  plates  was  injected  subcutaneously.     Animal  dead 
on  the  following  day. 


Hanging  drops  were  chiefly  relied  on  for  ascertaining  the 
reactions,  though  in  many  instances  these  were  controlled  by 
the  macroscopic  method  and  the  contents  of  the  tubes  examined 
microscopically  after  reaction  had  taken  place. 

The  sources  of  error  seem  about  equal  in  the  two  methods, 
while  the  hanging  drop  has  the  advantage  of  shorter  time  limit 
of  reaction,  and  of  easy  recognition  of  contamination. 

With  the  pneumococcus  the  tube  method  generally  indicates 
a  higher  microscopical  reaction  than  the  hanging  drop.  This 
is  contrary  to  tests  made  with  the  dysentery  and  typhoid  bacilli, 
and  is  explained  by  the  fact  that  in  the  former  case  the  free 
organisms  must  be  present  in  great  numbers  to  cloud  the  super- 
natant fluid,  whereas  in  the  latter  a  comparatively  small  number 
of  free  bacilli  may  render  the  fluid  turbid,  so  that  in  the  case 
of  the  pneumococcus  a  good  reaction  viewed  macroscopically 
may  become  only  a  fair  reaction  when  viewed  microscopically. 

3  A  description  of  this  organism  will  be  found  in  the   article  by  Drs.  Park 
and  Williams  in  this  volume. 


Katherine  E.  Collins 


21 


Neufeld  states  that  the  various  strains  of  the  pneumococcus 
agglutinate  alike,  an  observation  probably  due  to  the  low  re- 
actions obtained  by  him,  since  his  maximum  reaction  was  i :  50. 
My  work  has  shown  great  irregularity  in  this  respect,  the  serum 
of  an  animal  immunized  with  one  strain  of  pneumococcus 
agglutinating  only  seven  organisms  out  of  seventy  tested  in 
dilutions  equalling  the  reaction  (i :  200)  with  the  homologous 
organism.  Four  strains  reacted  in  dilutions  of  i :  10,  eleven  in 
1:2,  while  the  remaining  organisms  were  entirely  negative. 

The  serum  of  a  second  immunized  animal  agglutinated  the 
homologous  organism  in  dilution  of  i :  100,  while  other  strains 
were  affected  in  less  dilutions  or  not  at  all. 

TABLE  I. 

AGGLUTINATION  TESTS  WITH  THE  SERUM  OF  A  SHEEP*  INJECTED  FOR  A  PERIOD 
OF    THREE    MONTHS    WITH    PNEUMOCOCCUS    NO.    36. 


Dilution. 

2 

10 

20 

50 

100 

Control. 

Typical  pneumococcus  36 

+  + 

+  4- 

+  4- 

+  4- 

4-1 

— 

14 

— 

— 

— 

4 

+  + 

I 

I 

16 

+  + 

— 

— 

18 

— 

— 

33 

— 

I 

Atypical              "                 2 

+  + 

— 

— 

66 

+  1 

— 

I 

Pneumococcus  mucosus  47 

— 

— 

The  remaining  sixty-one  organisms  tested  reacted  some  in  dilution  of  i :  2  and 
Others  not  at  all,  thus  emphasizing  the  distinction  of  pneumococcus  No.  36 
from  the  other  strains  in  regard  to  its  power  to  produce  agglutinins. 


*  For  the  details  of  the  immunization  of  the  sheep,  the  paper  of    Drs.  Park 
and  Williams  is  to  be  consulted. 


22  Reaction  of  Agglutination  to  the  Pneumococcus 

There  is  apparently  a  difference  in  the  agglutinability  of  the 
pneumococci,  some  strains  uniformly  reacting  much  more  readily 
than  others  in  normal  and  immune  sera. 

A  rather  interesting  fact  that  deserves  further  investigation 
is  this:  Two  strains  of  pneumococci  which  showed  good  agglu- 
tination with  several  active  sera  failed  to  produce  agglutinins 
to  any  extent  in  the  animal  body  for  themselves  or  other  strains 
of  pneumococci.  A  rabbit  and  a  goat  were  inoculated  without 
results  with  one  strain,  and  a  rabbit  and  a  horse  with  the  other ; 
and  as  the  same  kind  of  animals  was  immunized  under  the  same 
conditions  with  other  strains  with  good  results,  this  irregularity 
would  seem  to  indicate  a  peculiarity  of  the  organism  rather 
than  of  the  animals  injected. 

Another  observation  of  interest,  but  one  which  has  not  been 
carried  far  enough  on  account  of  insufficient  time  to  estab- 
lish definite  conclusions,  is  certain  reactions  obtained  with 
a  streptococcus  serum  and  absorption  experiments  made  with 
this  organism, 

A  young  goat  which  was  immunized  with  a  strain  of  strepto- 
coccus yielded  a  serum  which  agglutinated  a  few  pneumococci 
and  its  own  culture  in  dilutions  of  i :  lo. 

Pneumococcus  66,  which  coagulates  inulin-serum  water  late, 
absorbs  the  agglutinins  for  several  pneumococci  from  a  typical 
pneumococcus  immune  serum.  This  culture  is  the  only  one  of 
the  pneumococci  that  has  its  agglutinins  taken  out  of  the  above 
streptococcus  imm-une  serum,  by  the  streptococcus ;  it  produces 
agglutinins  in  the  animal  body  for  itself  and  many  of  the  pneumo- 
cocci. These  reactions  suggest  the  possibility  of  the  occurrence 
of  intermediate  types  of  organisms  between  pneumococci  and 
streptococci. 

EXHAUSTION    EXPERIMENTS. 

The  extreme  sensitiveness  of  the  pneumococcus  to  changes  of 
conditions  not  readily  determined  brings  about  variation  in  the 
behavior  of  this  organism  which  proved  a  serious  factor  in  the 
application  of  the  agglutination  reaction  and  in  the  interpreta- 
tion of  the  results  obtained.     To  eliminate  as  far  as  possible 


Katherine  E.  Collins  23 

any  errors  arising  from  this  instability,  the  exhaustion  experi- 
ments were  conducted  in  groups,  each  group  covering  as  many 
observations  as  practical,  in  order  to  insure  uniform  conditions 
for  a  nimiber  of  tests. 

In  the  exhaustion  experiments  a  slight  loss  of  agglutinins  has 
generally  been  observed.  This  loss  occurs  whether  the  organism 
used  for  absorption  is  an  homologous  or  a  related  one  or  of  a 
foreign  type.  This  fact  points  to  the  cause  of  the  loss  lying 
outside  of  the  presence  of  the  organism.  The  loss  is  readity 
estimated  on  account  of  its  uniformity,  and  in  no  way  affects 
the  determination  of  the  amount  of  absorption  excepting  where 
a  strain  reacts  only  in  low  dilutions.  In  this  case  the  disap- 
pearance of  the  agglutinins  cannot  be  ascribed  with  certainty 
to  the  organism  used  for  absorption,  and  the  establishment  of 
relationship  by  the  absorption  method  is  not  possible  in  these 
instances. 

Testing  the  reaction  of  the  meningitis  coccus  in  antipneu- 
mococcus  serum,  Sorgente  failed  to  obtain  agglutination  with  a 
number  of  strains.  We  failed  to  absorb  the  agglutinins  from 
a  serum  agglutinating  several  strains  of  pneumococci  in  dilu- 
tion of  1 :  200  with  a  culture  of  the  diplococcus  of  meningitis. 

As  shown  in  Table  II,  the  power  of  the  serum  to  agglutinate 
pneumococcus  Nos.  14  and  72  in  equally  high  dilutions  with 
the  homologous  organism,  and  by  absorption  that  the  agglu- 
tinins are  group  agglutinins  in  the  case  of  the  former,  and  both 
group  and  specific  agglutinins  in  the  case  of  the  latter  organisms, 
are  readily  explained  by  the  fact  that  the  two  types  of  agglutinins 
constantly  vary  in  ratio  both  in  different  animals  and  at  different 
periods  of  inoculation,  the  group  agglutinins  exceeding  even 
at  times  the  specific  ones. 

The  increase  of  agglutinins  for  different  strains  of  Pneumo- 
coccus mucosus  in  the  serum  of  an  animal  inoculated  with  one 
of  the  typical  pneumococcus  strains,  and  the  results  obtained  by 
the  absorption  of  these  agglutinins,  separate  them  into  a  distinct 
variety  from  the  majority  of  other  pneumococci. 

The  similar  results  obtained,  as  indicated  in  the  following 
table,  by  absorption  with  Pneumococcus  mucosus  No.  47  and 


0 

z 

< 

a 

a 

o 

fe 

OT 

w 

P 

n 

X 

a 

U 

n 

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z 

a 

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2 

n 

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2 

w 

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o 

o 
o 
o 

O 

p* 

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a 

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pq 

CO 

Cii 

After  E.xhaustion 
with  D.  typhosus. 

o 
o 

+;+t    - 

o 

+++++++ 

o 

++++++++ 
++++++++ 

»o 

++++++++ 

l| 

o 
o 

r+tr 

o 
o 

u— + 

o 

++++ 1 +++ 

0 

++++++++ 
++++++++ 

o 

++++++++ 
++++++++ 

"'t'  'ONj  sno 

-ooooiunguj 

ii;iiVi  not; 

0 

1    1  '  '  1  ' 

o 

r-'it- 

«  o 
^  o  ■* 

'•<  ^  A 

o 
o 

»" 

0 

++++       1 

o 

+++++++„ 
+++++ 1  I 

o 

+  4-  +  +  +  '^  1    1 

■sdAx  n.u.0 
s^i  qiiAV  uon 

g 

1    1    1    ,  -r  1    1 

o 

1 ' ^1 +^+ 

d 
.2 

S 
"Pi 

H 
p 
B 

o 
H 

1 

a 
o 

1    1       i   "1    1    1 

M            H            H 

o 
-o 

++ ' +         ^^ 

o 
o 

++M+                   „+ 
++++                  ++ 

o 

+  +  +  +  ^'^  +  + 

o 

++++++++ 

+ 
1 

o 

++++++++ 
++++++++ 

+ 

o 

+++++++++ , 
++++++++ 1 

"            "          + 

N   "to  O         r~        MWt<2lOcy           • 

d    ^ 

. 

Pneumococcus 
mucosus. 

Atypical 
Pneumococci. 

Typical 
Pneumococci. 

Typical     r  Simple 
Pneumo-  J     Cold. 
cocci. 

.  Normal. 

-d     S 


^  c 


u     + 
CL,      + 


24 


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W    K    < 


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p   o 


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25 


26  Reaction  of  Agglutination  to  the  Pneumococcus 

pneumococcus  No.  4  (the  latter  organism  when  studied  not 
producing  the  characteristic  capsule  and  gelatinous  colonies), 
suggest  that  these  cultural  attributes  may  be  lost  while  the 
agglutinative  affinity  is  still  retained. 

The  ability  of  the  Pneumococcus  mucosus  group  to  produce 
common  agglutinins  for  some  pneumococci,  and  the  fact  that  the 
streptococcus  failed  to  affect  through  absorption  their  agglutinins, 
would  indicate  a  closer  relation  of  this  variety  to  the  pneumococci 
than  to  the  streptococci. 

CONCLUSIONS, 

Owing  to  unavoidable  circumstances  only  a  limited  amount 
of  time  was  available  for  the  work  on  agglutination.  The 
foregoing  report  is  therefore  preliminary  only  and  the  following 
conclusions  are  provisionally  offered : 

I.  Pneinnococci  by  reason  of  their  agglutinating  properties 
exhibit  a  tendency  to  separate  into  numerous  groups  similar  to 
streptococci. 

II.  Pneumococcus  mucosus  forms  a  distinct  and  consistent 
variety.  The  production  by  it  of  common  agglutinins  for  some 
pneumococci  and  the  resistance  of  the  agglutinins  produced  by 
it  to  absorption  by  the  streptococcus  indicate  a  nearer  relation 
to  the  former  than  to  the  latter  organism. 

III.  The  agglutinating  substances  in  the  serum  of  immunized 
animals  were  demonstrated  by  absorption  tests  to  consist  of 
specific  and  group  agglutinins  in  cases  where  the  agglutinins 
were  sufficiently  developed  to  make  use  of  this  method, 

IV.  The  pneumococci  seem  to  show  marked  differences  in 
their  ability  to  undergo  agglutination. 

V.  There  was  considerable  uniformity  of  reaction  of  the 
various  strains  in  low  dilutions,  but  this  uniformity  is  not  con- 
tinued as  the  animal  becomes  more  highly  immunized. 

VI.  At  present  it  is  not  possible  to  establish  a  definite  rela- 
tion between  the  agglutination  reaction  and  the  other  char- 
acteristics of  the  pneumococcus  excepting  in  the  case  of  the 
Pneumococcus  mucosus. 


Katherine  E.  Collins  27 

The  above  work  has  been  conducted  under  the  direction  of  Dr. 
Wm.  H.  Park,  Director  of  the  Research  Laboratory  of  the 
Department  of  Health  of  New  York  City,  to  whom  I  desire  to 
express  my  appreciation  of  the  interest  he  has  shown  in  the 
work. 

REFERENCES. 

Bolduan. — New  York  Medical  Journal,  1905,  Ixxxi,  956. 

Clairmont. — Zeitschrift  filr  Hygiene  und  I nfektionskrankheiten,-  1902,  xxxix,  i. 
Heyrovsky. — Centralblatt   f.  Bakteriologie  u.  Parasitenkunde,   1905,  xxxviii, 
704. 

Hiss. — Journal  of  Experiinental  Medicine,  1905,  vii,  223. 

Landsteiner. — Wiener  klinische  Wochenschrip,  1897,  x,  439. 

Morello. — Annali  d'Igiene  Sperimentale,  1904,  xiv,  153. 

Neufeld. — Zeitschrift  fur  Hygiene  und  Infektionskrankheiten,  1902,  xl,  54. 

Park  and  Collins. — Jotirnal  of  Medical  Research,  1904,  vii,  491. 

Sorgente. — Centralblatt  f.  Bakteriologie  u.  Parasitenkunde,  1905,  xxxix,  i. 

Wadsworth. — Journal  of  Medical  Research,  1903,  x,  228. 


A  CO^IPARATIVE  STUDY  OF  PXEUMOCOCCI  AND 
STREPTOCOCCI  FROM  THE  MOUTHS  OF  HEALTHY 
INDIVIDUALS  AND  FROM  PATHOLOGICAL  CON- 
DITIONS. 

By  WARFIELD  T.   LOXGCOPE,  M.D., 

Director  of  the  Ayer  Clinical  Laboratory  of  the  Pennsylvania  Hospital, 

Philadelphia, 

AXD  W.  W.  FOX,  M.D. 

From  the  ist  of  November,  1904,  until  the  ist  of  May,  1905, 
there  were  studied  sixty-nine  strains  of  organisms  which  ap- 
peared to  belong  to  the  groups  of  streptococci  and  pneumococci. 
The  morphological,  biological,  and  pathogenic  properties  of  these 
organisms  were  investigated.  For  convenience  of  description 
and  study  all  these  bacteria  were  grouped  into  three  main 
classes,  Series  A,  Series  B,  and  Series  C. 

Series  A  comprised  all  the  organisms  isolated  from  the  saliva 
of  healthy  individuals,  by  the  inoculation  of  the  saliva  into  mice 
and  rabbits,  by  making  streak  plates  from  the  buccal  secretion, 
or  by  both  methods. 

Series  B  included  all  the  organisms  obtained  from  pathological 
conditions,  which  from  their  cultural  characteristics  and  effects 
upon  animals  were  thought  to  be  pneumoccoci. 

Series  C  included  all  the  organisms  obtained  from  pathological 
conditions  which  from  appropriate  tests  were  considered  as 
streptococci. 

METHODS. 

In  general  the  method  of  procedure  was  to  study  the  mor- 
phology of  the  organisms  by  cover-slip  preparations  from  the 
original  fluid  or  organ  from  which  they  were  cultivated.  Three 
varieties  of  culture  medium  at  least  were  employed — blood-agar, 
plain  agar,  and  blood-serum, — and  the  morpholog}^  was  studied  in 
the  cultures  and  the  exudates  and  body  fluids  of  the  inoculated 

28 


Warfield  T.  Longcope  and  W.  W.  Fox 


29 


animals .  As  a  routine  the  organisms  were  grown  upon  blood-agar , 
plain  agar,  bouillon,  litmus  milk,  inulin-senmi  water,  potato, 
gelatine,  and  imheated  human  blood-serum.  The  media  were 
all  standardized  by  titration  with  ^V  N.  sodium  hydroxide  and 
made  neutral  to  phenolphthaleine.  It  may  be  said  that  the 
onl}^  media  which  proved  of  great  practical  value  in  differentiat- 
ing the  various  strains  of  cocci  were  inulin-serum  water,  blood- 
serum,  and  blood-agar.  The  blood-agar  was  of  great  service  as 
a  mediimi  for  preserving  stock  cultures.  It  was  made  by  the 
addition  of  a  few  drops  of  defibrinated  human  blood  to  a  tube  of 
melted  agar.  Organisms  grown  upon  this  medium,  in  tubes 
covered  with  a  rubber  cap,  remained  viable  for  several  weeks 
when  kept  in  the  dark  at  o°  to  io°  C.  Moreover,  their  virulence 
was  well  retained.  One  culture  of  Series  B  of  pneumococci  after 
a  month's  grouch  on  blood-agar  killed  rabbits  in  doses  of  .0001 
c.c.  of  a  twenty-four-hour  bouillon  culture,  which  was  the  original 
minimal  fatal  dose. 

Finally,  the  virulence  of  the  organisms  was  tested  for  rabbits 
and  mice,  and  the  lesions  produced  in  these  animals  studied 
bacteriologically  and  frequently  histologically. 

Series  B  may  first  be  considered;  it  comprises  sixteen  or- 
ganisms. The  following  table  gives  the  main  data  concerning 
their  growth  and  pathogenic  properties. 

TABLE  I. 
Series  B. 


No.  of 
Organism. 

Source. 

Date. 

Morphologj-. 

Capsules. 

Inulin- 
Serum 
Water. 

Pathogenicity. 

IL 

Blood  cul- 
tiire,  lobar 
pneu- 
monia. 

I 1/4/04 

Lanceolate 
diplococci, 
no  chains. 
Gram 
positive. 

Positive ; 

blood-agar. 

48  hrs., 
clot. 

0.5  C.C.  24-hr.  bouillon  culture 
subcutaneously  does  not  kill 
mice.     I  c.c.  24-hr.  bouillon 
culture  subcutaneously  kills 
rabbit  ,760  grams,  in  1 8  days ; 
much   emaciation;   no   local 
lesions.    Cultures  from  heart 
and  peritoneum  sterile.    One 
blood-agar    slant    intraperi- 
toneally     kills     rabbit,     wt. 
1020  grams,  in  36  hrs.     Fi- 
brino-purulent      peritonitis; 
large  hard  spleen;  cultures, 
heart  and  peritoneum,  posi- 
tive. 

30  Comparative  Study  of  Pneumococci  and  Streptococci 

TABLE  I  {Continued). 


No.  of 
Organism. 


III. 


IV. 


V. 


VIII. 


Source. 


Cultiire 
from  con- 
solidated 
lung,  lobar 
pneu- 
monia. 


Meninges, 
cerebro- 
spinal 
menin- 
gitis. 


Date. 


11/6 


Pleiira, 
lobar 
pneu- 
monia. 


Lung, 
lobar 
pneu- 
monia. 


1 1/8 


ii/iS 


12/5 


Morphology. 


Lanceolate 
diplococci, 
no  chains. 
Gram 
positive. 


Lanceolate 
diplococci, 
no  chains. 
Gram 
positive. 


Lanceolate 
diplococci, 
few  short 
chains. 
Gram 
positive. 


Lanceolate 
and  round 
cocci  in 

pairs.' 

Gram'" 

positive. 


Capsules. 


Blood-agar; 
positive 
(ordinary 
stains). 


Blood-agar ; 
positive 
(ordinary 
stains). 


Inulin- 
Serum 
Water. 


24  hrs., 
clot. 


Positive ; 
serum 
(Hiss). 


48  hrs. 
clot. 


48  hrs., 
clot. 


48  hrs. 
clot. 


Pathogenicity. 


4  c.c.  24-hr.  bouillon  cultun 
subcutaneously  kills  rabbit 
wt.  708  grams,  in  5  days 
Extensive  sero-fibrinous  sub, 
cutaneous  exudate ;  broncho 
pneumonia ;  spleen  large  an( 
soft.  Cultures  from  hear 
positive.  2  c.c  24-hr.  bouilloi 
culture  kills  mouse  intra 
peritoneally  in  12  hrs. 

2  c.c.  24-hr.  bouillon  cultur 
intravenously  kills  rabbit 
■wt.  374  grams,  in  12  hrs 
Spleen  small  and  hard.  Afte 
passage  through  13  rabbits 
o.ooi  c.c.  24-hr.  bouillo: 
culture  kills  rabbit,  \\A,.  195 
grams,  in  36  hrs.;  produce 
usually  extensive  fibrinou 
exudate  in  subcutaneous  tis 
sues  with  hard  friabl 
spleen,  also  fibrino-purulen 
peritonitis,  septicemia  wit 
subserous  haemorrhages ;  one 
infarction  of  spleen. 

2  c.c.  24-hr.  bouillon  cultm 
subcutaneously,  in  rabbit, w 
1300  grams,  negative.  2  c.i 
24-hr.  bouillon  culture  ii 
traperitoneally,  in  rabbit, w 
1320  grams,  negative.  Deat 
in  35  da3^s,  no  local  lesioni 
cultures  negative. 

0.2  c.c.  24-hr.  bouillon  cu 
ture  subcutaneously  kil 
rabbit,  wX.  1000  grams,  i 
12  hrs.  Extensive  sero-sai 
guino-fibrinous  exudate  \ 
subcutaneous  tissues;  sma 
soft  spleen.  Cultures  an 
cover-slips  from  heart,  su" 
cutaneous  tissues,  and  pei 
toneum  positive. 

0.5  c.c.  24-hr.  bouillon  cultu 
kills  second  rabbit,  wt.  15: 
grams,  subcutaneously, 
20  hrs.  Slight  subcutaneoi 
oedema,  small  soft  splee: 
cultures  from  subcutaneoi 
tissues,  heart,  and  perit 
neum  positive. 


Warfield  T.  Longcope  and  W.  W.  Fox                     31 

TABLE  I   {Continued). 

No.  of 
Organism. 

Source. 

Date. 

Morphology. 

Capsules. 

Inulin- 
Serum 
Water. 

48  hrs., 

Pathogenicity. 

IX. 

Pus,  em- 

12/10 

Lanceolate 

Positive ; 

0.4  c.c.  24-hr.  bouillon  culture 

pysema. 

and 

rounded 

cocci  in 

pairs. 

Gram 

positive. 

serum ; 

blood-agar 

(Hiss). 

clot. 

subcutaneously,  in  rabbit, 
wt.  1480  grams,  death  in  12 
days ;  extensive  fibrinous 
exudate  in  subcutaneous  tis- 
sues; large,  hard  spleen. 
Cultures  from  heart  positive. 

X. 

Pus,  em- 

12/13 

Lanceolate 

Positive ; 

No  clot, 

2  c.c.   24-hr.  bouillon  culttire 

pyaema. 

cocci  in 
pairs. 
Gram 
positive. 

serum 
(Hiss). 

10  days. 

subcutaneously  in  rabbit, 
wt.  1 100  grams,  death  in  36 
hrs.  Extensive  sero-fibrin- 
ous  exudate,  small  soft 
spleen;  culttires  and  cover- 
slips  positive.  From  sub- 
cutaneous tissues  and  heart, 
capsules  readily  stainable. 

One  24-hr.  blood-agar  culture 
subcutaneously  in  rabbit, 
wt.  1900  grams,  death  3  days 
12  hrs.  Same  lesions  as 
above,  with  sero-fibrinous 
peritonitis. 

One  24-hr.  blood-agar  culture 

XI. 

Lung, 

12/17 

Lanceolate 

Positive ; 

48  hrs., 

lobar 

cocci  in 

serum- 

clot. 

subcutaneously     in     rabbit. 

pneu- 

pairs. 

(Hiss). 

wt.   1320  grams.      Lives. 

monia. 

Gram 

positive. 

XII. 

Otitis 

1/14 

Lanceolate 

Positive ; 

48  hrs., 

One  24-hr.  blood-agar  culture 

media. 

cocci  in 

pairs  and 

short 

chains. 

Gram 

positive. 

Blood- 
agar 

(ordinary 
stains). 

clot. 

subcutaneously  in  rabbit, 
wt.  820  grams;  death  in  7 
days.  Localized  subcutane- 
ous fibrinous  exudate,  fibrin- 
ous peritonitis,  and  pericar- 
ditis. Cultures  from  heart, 
peritoneum,  and  pericardium 
positive.  Rabbit  3.  o.i  c.c. 
24-hr.  bouillon  culture  kills 
in  6  days.  Cultures  from 
heart  and  peritoneum  posi- 
tive. 

XIII. 

Lung, 

1/19 

Lanceolate 

Positive ; 

48  hrs., 

One    24-hr.    blood-agar   slant 

broncho- 

cocci in 

serum 

clot. 

subcutaneously     in     rabbit, 

pneu- 

pairs. 

(Hiss). 

wt.    1400   grams;     death   in 

monia. 

Gram 
positive. 

10  days.  Fibrinous  subcu- 
taneous exudate.  Cultures 
from  heart  negative. 

32  Comparative  Study  of  Pneumococci  and  Streptococci 

TABLE  I  {Continued). 


No.  of 
Organism. 


XIV. 


XVI. 


XVII. 


XVIII. 


Source. 


Pus,  em- 
pyasma. 


Retro- 
pharyn- 
geal 


Spinal 
fluid, 
menin- 
gitis. 


Date. 


1/30 


XIX. 


Lung  punc- 
ture, lobar 
pneu- 
monia. 


Sputum; 
same  case 
as  XVIII; 
taken  at 
same  time. 


1/30 


2/20 


3/S 


3/5 


Morphology. 


Lanceolate 
cocci  in 
pairs  and 
short 
chains. 


Round  and 
lanceolate 
cocci  in 
pairs. 
Gram 
positive. 

Lanceolate 
cocci  in 
pairs  and 
short 
chains. 
Gram 
positive. 


Lanceolate 
and  oval 
cocci  in 
pairs  and 
long 
chains. 
Gram 
positive. 


Lanceolate 
and  round 
cocci  in 
pairs. 
Gram 
positive. 


Capsules. 


Positive ; 
serum 
(Hiss). 


Positive ; 
serum 
(ordinary 
stains). 


Positive ; 
serum 
(Hiss). 


Inulin- 
Serum 
Water. 


24  hrs. 
clot. 


72  hrs. 
clot. 


48  hrs., 
clot. 


Positive ; 
serum 
(Hiss). 


Positive ; 
serum 
(Hiss). 


48  hrs., 
clot. 


48  hrs., 
clot. 


Pathogenicity. 


O.I  c.c.  24-hr.  bouillon  culture 
subcutaneously  in  rabbit, 
wt.  1500  grams;  death  in  2 
days  12  hrs.  Sero-purulent, 
peritonitis,  pleuris}'-,  and 
pericarditis.  Small     soft 

spleen.  Cultures  from  heart 
positive.  After  passage  in 
decreasing  doses  through  10 
rabbits,  0.000,001  c.c.  36-hr. 
bouillon  culture  intraperi- 
toneally  in  rabbit,  wt.  1220 
grams;  death  in  12  hrs.  Lei 
sions,  sero-purulent  exudata 
in  serous  cavities  as  a  rul6s 
spleen  sometimes  hard, 
sometimes  soft;  slight  sub- 
cutaneous oedema ;  septi- 
csemia  always. 

One  24-hr.  blood-agar  slant 
subcutaneously  in  rabbit, 
vrt.  988  grams;  death  in  19 
days.  Slight  subcutaneo- 
fibrinous  exudate. 


I  c.c.  24-hr.  bouillon  culture 
subcutaneously  in  rabbit, 
wt.  580  grams;  death  in  3 
days.  Sero-fibrinous  exu- 
date in  subcutaneous  tissues. 
Cultures  from  heart  and  sub- 
cutaneous tissues  positive. 
3d  rabbit.  0.0 1  c.c.  24-lir. 
bouillon  culture  subcuta- 
neously, wt.  822  grams; 
death  in  8  days.  Sero- 
purulent  peritonitis ;  cul- 
ture negative. 

I  c.c.  24-hr.  bouillon  culture 
subcutaneously  in  rabbit, 
wt.  1290  grams;  death,  6 
days.  Subcutaneous  fibrin- 
ous exudate,  spleen  hard, 
sero-fibrinous  peritonitis. 
Cultures  from  heart  and 
peritoneum  positive.  0.5 
c.c.  24-hr.  bouillon  culture 
subcutaneously  does  not  kill 
rabbits. 

I  c.c.  24-hr.  bouillon  culture 
subcutaneously  in  rabbit, 
wt.  860  grams;  death  in  36 
hrs.  Subcutaneous  conges- 
tion, oedema,  and  haemor- 
rhage; large,  hard  spleen. 
Cultures  from  heart  positive. 


Warfield  T.  Longcope  and  W.  W.  Fox 
TABLE  I  (Continued). 


33 


No.  of 
Drganism. 

Source. 

Date. 

Mordhology. 

Capsules. 

Inulin- 
Serum 
Water. 

Pathogenicity. 

XX. 

Heart  vege- 
tations ; 
acute 
endocar- 
ditis. 

3/30 

Lanceolate 
and  oval 
cocci  in 
pairs. 

Positive ; 
serum 
(Hiss). 

48  hrs., 
clot. 

0.5   c.c.    24-h.r.    bouillon   cul- 
ture intraperitoneally  in  2d 
rabbit,     wt.      1050     grams; 
death  in  12  hrs.      Moderate 
fibrino-purulent    peritonitis ; 
large,  hard  spleen.    Cultures 
from  heart  and  peritoneum 
positive. 

The  morphology  was  usually  that  of  lanceolate  diplococci; 
this  was  most  constant  in  blood-agar,  in  serum,  and  in  the 
exudates  of  animals.  In  other  media  chains  of  cocci  were 
frequently  noted,  some  made  up  of  oval,  almost  round,  or  even 
slightly  flattened  elements.  All  stained  positively  by  Gram's 
method  of  staining.  Capsules  cotdd  frequently  be  demonstrated 
by  ordinary  stains  on  organisms  grown  in  serum,  and  without 
fail  by  the  stains  of  Hiss.  On  blood-agar  the  growth  was  quite 
luxuriant,  moist,  raised,  grayish,  and  often  formed  a  thin  watery 
film.  There  was  always  a  marked  tendency  for  the  colonies  to 
become  confluent.  The  growth  was  heaviest  and  most  typical 
with  freshly  isolated  organisms.  In  blood-serum  a  very  heavy 
cloud  appeared  in  twenty-four  to  forty-eight  hours,  and  large 
quantities  of  acid  were  formed.  The  serum  in  all  these  routine 
tests  was  taken  from  one  patient  suffering  from  chronic  endo- 
carditis who  was  bled  copiously.  In  inulin-serum  water  all  the 
organisms  except  one  (lo  B)  produced  acid  and  formed  a  solid 
clot  in  from  twenty -four  to  seventy-two  hours.  This  one  or- 
ganism produced  perhaps  faint  traces  of  acid  but  no  clot,  even 
after  ten  days'  growth.  In  all  other  respects  it  was  identical 
with  the  other  cocci  of  this  group  and  unlike  those  grouped  in 
Series  C. 

The  pathogenicity  of  these  bacteria  varied  greatly,  from 
strains  which  even  in  large  doses  produced  almost  no  effect  in 
rabbits  to  strains  which  were  highly  virulent.  No  relationship 
could  be  traced  between  the  source  of  the  culture  and  the  degree 


34  Comparative  Study  of  Pneumococci  and  Streptococci 

of  virulence.  The  most  virulent  cultures  were  obtained  from 
pus  from  cases  of  empyeema,  and  from  the  cerebro-spinal  fluid 
of  cases  of  meningitis.  Most  of  the  cultures  from  the  consoli- 
dated lungs  of  cases  of  lobar  pneumonia  showed  a  rather  low 
grade  of  virulence.  In  five  instances  emulsions  of  consolidated 
lungs  were  inoculated  into  animals.  The  animals  either  did  not 
die  or  lived  for  many  days,  and  at  autopsy  cultures  from  the 
organs  gave  no  results.  In  one  instance  (B  III),  cultures  directly 
from  the  lung  yielded  a  pneumococcus  which  in  large  doses  killed 
rabbits.  In  two  other  instances  streptococci  (C  VII  and  C  XI) 
were  recovered  in  cultures  from  the  lungs.  Of  two  organisms  (B 
XVIII  and  B  XIX)  obtained  in  one  instance  from  the  saliva  and 
from  lung  puncture  in  the  same  person  suffering  from  acute  lobar 
pneumonia,  the  coccus  from  the  saliva  proved  the  more  virulent 
for  rabbits.  The  pathological  processes  produced  in  the  various 
animals  varied  greatly;  no  one  organism  gave  a  constant  type 
of  lesion.  Extensive  subcutaneous  exudates  of  fibrin,  fibrin  and 
pus,  and  fibrin  and  serum  were  common.  Fibrino-purulent 
peritonitis,  pleurisy,  pericarditis,  and  mediastinitis  were  often 
seen;  septicaemia  with  and  without  hemorrhages  in  the  serous 
membranes  and  thymus  gland  were  noted.  In  most  instances  the 
spleen  was  hard,  friable,  and  showed  on  microscopic  examination 
an  extensive  infiltration  of  fibrin  with  hyaline  thrombi  in  the 
blood  spaces.  Occasionally  it  was  soft  and  red.  Both  con- 
ditions were  noted  in  animals  inoculated  at  different  times  with 
the  same  organism.  The  liver  was  sometimes  fatty,  often 
showed  cloudy  swelling  and  congestion,  and  in  some  instances 
presented  on  microscopic  examination  foci  of  necrosis  infiltrated 
with  fibrin  and  leucocytes.  The  kidneys  were  often  congested, 
and  in  some  cases  hyaline  thrombi  were  observed  in  the  glomeru- 
lar capillaries.  In  one  instance  these  had  much  the  appearance 
of  agglutinated  red-blood-corpuscle  thrombi.  The  heart  was 
often  flabby  and  distended  with  blood.  The  lungs  were  usually 
congested.  In  no  instance  was  a  pneumonia  seen  which  could 
be  ascribed  to  the  action  of  the  pneumococcus.  A  few  animals 
became  gradually  emaciated  and  died  after  some  weeks  without 
local  lesions. ^^t'  At  autopsy  cultures  from  the  organs  gave  negative 


Wariield  T.  Longcope  and  W.   W.  I" 

OX                    35 

results.     In 

L  a  few  instances  the  animals  developed  sloughing 

sores  of  the  abdominal  wall  from  which  they  died  after  several 

days.     Cultures  usually  showed  a  secondary  invasion  by  another 

organism. 

If  these  findings  are  compared  with  the  table  below,  it  will  be 

seen  how  different  are  the  two  groups  B  and  C. 

TABLE  II. 

Series  C. 

Inulin- 

No.  of 

Source. 

Date. 

Morphology. 

Capsules. 

Serum 

Pathogenicity. 

rganism. 

Water. 

I. 

Blood  cul- 

11/9 

Small, 

Negative ; 

No  clot. 

Not  tried. 

ture,  mas- 

round 

serum 

6  days. 

toiditis. 

cocci  in 

clusters 

and 

chains. 

Gram 

positive. 

(Hiss). 

II. 

Arthritis, 

11/14 

Round  and 

Negative ; 

No  clot. 

Not  tried. 

pus  from 

slightly 

serum 

7  days. 

joint. 

lanceolate 
cocci  in 
pairs  and 
long 
chains. 
Gram 
positive. 
Round 

(Hiss). 

III. 

Peritonitis, 

11/21 

Negative ; 

No  clot. 

2   c.c.    24-hr.    blood    bouillon 

case  of 

cocci  sin- 

serum 

8  days. 

culture  intraperitoneally  in 

typhoid 

gly,  in 

(Hiss). 

rabbit,     wt.      1050     grams; 

fever  com- 

pairs, and 

death  in  21  days.    Arthritis, 

plicated 

chains. 

left  hind  paw.   Cultures  con- 

with lobar 

Gram 

taminated. 

pneu- 

positive. 

monia. 

IV. 

Blood  cul- 

11/25 

Small, 

Negative ; 

No  clot. 

Not  tried. 

ture,  puer- 

round 

serum 

6  days. 

peral 

cocci  in 

(Hiss). 

sepsis. 

pairs  and 
chains. 
Gram 
positive. 

V. 

Lung,  acute 

11/28 

Small, 

Negative ; 

No  clot, 

Not  tried. 

broncho- 

round 

serum 

7  days. 

pneu- 

cocci sin- 

(Hiss). 

monia. 

gly  and  in 
pairs. 
Gram 
positive. 

36  Comparative  Study  of  Pneumococci  and  Streptococci 

TABLE  II   (Continued). 


No.  of 

Inulin- 

Organism. 

Soiirce. 

Date. 

Morphology. 

Capsules. 

Serum 
Water. 

Pathogenicity. 

VI. 

Pus  from 

12/31 

Small, 

Negative ; 

No  clot, 

Not  tried. 

abscess. 

round 
cocci  in 

pairs  and 
chains. 

serum 
(Hiss). 

7  days. 

VII. 

Lung, 

1/7 

Small, 

Negative ; 

No  clot. 

I   c.c.  emulsion   of  lung  sut? 

lobar 

round, 

serum 

6  days. 

cutaneously   in    rabbit,    wt. 

pneu- 

and lan- 

(Hiss). 

1260    grams;     death    in    14 

monia. 

ceolate 
cocci,  sin- 
gly, in 
pairs,  and 
short 
chains. 

days.  Subcutaneous  abscess. 
One  24-hr.  blood-agar  slant 
subcutaneously  in  rabbit, 
wt.  1090  grams;  death,  21 
days.  Large  circumscribed 
subcutaneous  abscess.  Cul- 
ture from  abscess  positive. 

VIII. 

Cultiu-e 

i/ii 

Small, 

Negative ; 

No  clot, 

Not  tried. 

from  lung. 

cocci  in 

serum 

6  days. 

m 

terminal 

chains  and 

(Hiss). 

1 

pneu- 

clusters. 

1 

monia. 

Gram 
positive. 

f 

IX. 

Lung, 

3/29 

Small, 

Negative ; 

No  clot. 

One    24-hr.    blood-agar    slant 

lobar 

round. 

serum 

7  days. 

intraperitoneally   in    rabbit, 

pneu- 

and flat- 

(Hiss). 

800  grams;  death  in  24  days. 

monia. 

tened  cocci 

in  pairs 

and 

chains. 

Gram 

positive. 

Death  due  to  septicaemia. 

X. 

Blood  cul- 

4/13 

Small, 

Negative ; 

No  clot, 

5  c.c.  original  24-hr.  bouillon 

ture,  em- 

round. 

serum 

7  days. 

culture   intraperitoneally  in 

pyema. 

and 

slightly 

lanceolate 

diplococci 

in  pairs 

and 

chains. 

Gram 

positive. 

(Hiss). 

rabbit.     Lived.                     1 

XI. 

Lung, 

4/20 

Small, 

Negative ; 

No  clot. 

2   c.c.   emulsion  of  lung  sub- 

broncho- 

round, and 

serum 

10  days. 

cutaneously    in    rabbit,    vd. 

pneu- 

lanceolate 

(Hiss). 

260   grams;    death,    12   hrs. 

monia. 

cocci  in 
pairs  and 
short 
chains. 
Gram  im- 
perfectly 
positive. 

Extensive  fibrino-purulent 
peritonitis.  Culture  gave 
C  XI.  Two  24-hr.  blood- 
agar  slants  subcutaneously 
in  rabbits.      Lived. 

Warfiekl  T.  Longcope  and  W.  W.  Fox 


37 


In  this  group,  Series  C,  there  are  eleven  organisms.  All  of 
them  were  cocci  occiirring  in  pairs  and  chains.  They  were 
round,  flattened,  or  appeared  slightly  lancet-shaped.  Gram 
stain  was  always  positive.  Capsules  could  never  be  demon- 
strated b}^  the  methods  employed.  On  blood-agar  the  growth 
was  a  dryish  film  composed  of  innumerable  minute  colonies 
which  gave  the  surface  a  granular  appearance ;  after  some  days' 
growth  the  blood  was  frequently  hasmolysed.  In  serum  there 
was  no  cloud  but  a  granular  sediment,  and  as  a  rule  there  was  no 
acid  produced.  Rarely  the  fluid  became  neutral  or  gave  litmus 
a  faint  reddish  tinge.  There  was  nothing  to  compare  with  the 
acid  production  seen  in  group  B.  Inulin-serum  water  was  never 
acidified  or  clotted.  The  organisms  rarely  killed  rabbits,  and  in 
the  animals  which  died  definite  lesions  were  often  missed.  Once 
an  arthritis  developed. 

Finally,  we  have  Series  A,  the  organisms  obtained  from  the 
mouths  of  healthy  individuals.  In  this  series,  as  shown  in  the 
following  tables,  there  are  forty- two  organisms.  These  cocci 
were  distinguishable  into  two  types,  to  be  called  Type  I  and 
Type  II. 

TABLE  in. 
Series  A.     Type  I. 


No.  of 
rganism. 

Source. 

Date. 

Morphology. 

Capsules. 

Inulin- 
Serum 
Water. 

Pathogenicity. 

L 

Hospital 
resident. 

1 1/2 

Lanceolate 
diplococci, 

few 
chains. 
Gram 
positive. 

Positive ; 
serum 
(Hiss). 

48  hrs., 
clot. 

I  c.c.  24-hr.  bouillon  culture 
subcutaneously     in    rabbit, 
wt.  330  grams;    death  in  36 
hrs.     Extensive    sero-fibrin- 
ous    subcutaneous   exudate; 
small,  hard  spleen.    Cultures 
from  heart's  blood  and  sub- 
cutaneous exudate  positive. 
The    organism    was    passed 
through   18  rabbits;    fibrin- 
ous   subcutaneous    exudate 
and     fibrino-purulent     peri- 
tonitis   were    the    usual    le- 
sions;    in   several   instances 
sero  -  fibrinous      pericarditis 
and   double  pleurisy.      Once 
extensive     fibrino  -  purulent 
mediastinitis.       Spleen  usu- 
ally hard   and  large,  some- 
times soft  and  small.      Rab- 
bit    19,     wt.      1290    grams. 
0.05  c.c.  24-hr.  bouillon  cul- 
ture intraperitoneally ;  death 
in  33  days.     Pneumococcus 
septicaemia. 

38  Comparative  Stvxly  of  Pneumococci  and  Streptococci 

TABLE  III  {Continued). 


No.  of 
Organism. 


VII. 


Source. 


Laboratory 
janitor. 


Date. 


4/14 


VIII.    Laboratory 
assistant. 


X. 


Hospital 
resident. 


XIII. 


Outdoor 
physician. 


XIV. 


XV. 


XVI. 


Hospital 
resident. 


11/26 


11/29 


12/1 


12/6 


Hospital 
resident. 


Laboratory 
assistant. 
Same  or- 
ganisms in 
plates 
from 
sputum. 


12/6 


12/6 


Morphology. 


Lanceolate 
cocci  in 
pairs. 
Gram 
positive. 


Lanceolate 
cocci  in 
pairs. 
Gram 
positive. 


Small 
lanceolate 
cocci  in 
pairs  and 
short 
chains. 
Gram 
positive. 

Lanceolate 

and 
round 
cocci  in 
pairs  and 
short 
chains. 
Gram 
positive. 


Lanceolate 
and  round 
cocci  in 
pairs. 
Gram 
positive. 


Lanceolate 
diplococci, 
no  chains. 
Gram 
positive. 


Round  and 
lanceolate 
cocci  in 
pairs  and 
short 
chains. 
Gram 
positive. 


Capsules. 


Positive ; 
blood-agar 
(Hiss). 


Positive ; 

blood-agar 

(Hiss). 
Positive ; 

serum 
(ordinary 

stain). 
Positive; 

serum 

(Hiss). 


Positive ; 
serum 
(Hiss). 


Positive ; 
serum 
(Hiss). 


Positive ; 
serum 
(Hiss). 


Positive ; 
serum 
(ordinary 
stains). 


Inulin- 
Serum 
Water. 


9  days, 
acid, 
thick- 
ening ; 
no  firm 
clot. 


24  hrs., 
clot. 


48  hrs. 
clot. 


24  hrs. 
clot. 


24  hrs. 
clot. 


4  days, 
clot. 


4  days, 
clot. 


Pathogenicity. 


Three  24-hr.  blood-agar  slants 
subcutaneously  in  rabbit, 
wt.  790  grams;  death  in  7 
days.  Extensive  subcutane- 
ous fibrinous  exudate;  ulcer- 
ation of  skin;  spleen  soft. 
Cultures  from  heart  and  sub- 
cutaneous tissues  positive. 

One  24-hr.  blood-agar  culture 
intraperitoneally  in  rabbits; 
lived.  Small  doses  have  no 
effect. 


2  c.c.  24-hr.  bouillon  culture 
subcutaneously  in  rabbit,  wt. 
900  grams.      Lived.  -  1 

1  c.c.  24-hr.  bouillon  culttire 
subcutaneously  in  mouse;' 
death  in  3  6  hours.  Extensive 
sero-fibrinous  exudate.  Cul- 
tures from  peritoneum  and 
heart  positive. 

2  c.c.  24-hr.  bouillon  culture 
subcutaneously  in  rabbit, 
•u^t.  980  grams;  death  in  28 
days.  No  local  lesions;  gen- 
eral septiccemia.  Cultures 
from  heart  positive. 

I  c.c.  24-hr.  bouillon  culttire 
intraperitoneally  in  mouse; 
death  in  12  hrs.  Cultures 
from  heart  and  peritoneum 
positive. 

1  c.c.  24-hr.  bouillon  culture 
subcutaneously  in  mouse; 
death  in  60  hrs.  Extensive 
sero-fibrinous  exudate.  Cul- 
tures from  subcutaneous  tis- 
sues and  heart  positive. 

2  c.c.  24-hr.  bouillon  culture 
subcutaneously  in  rabbit, 
wt.  1250  grams.      Lived. 

2  c.c.  24-hr.  bouillon  culture 
subcutaneously  in  rabbit, 
wt.  1 190  grams;  death  in  36 
hrs.  Sero-sanguineous  svjo- 
cutaneous  exudate,  acute 
sero-purulent  peritonitis. 
Culture  from  heart  positive. 

4  c.c.  24-hr.  bouillon  culture 
intraperitoneally  in  rabbit. 
Lived. 

Two  24-hr.  blood-agar  cul- 
tures intraperitoneally  in 
rabbit;  death  in  6  days. 
Slight  fibrinous  exudate 
in  subcutaneous  tissues, 
fibrino-purulent  peritonitis; 
large,  soft  spleen.  Cultures 
from  heart  and  peritoneum 
positive. 


Warfield  T.  Longcope  and  W.  W.  Fox 
TABLE  III   {Continued). 


39 


Xo.  of 
rganism. 


XVII. 


Source. 


Hospital 
resident. 
Same 
organism 
from 
saliva  in 
plates. 


XVIII. 


Date. 


12/13 


Laboratory 
assistant. 
Same 
organism 
from 
saliva  in 
plates. 


XIX.    Workman; 
open  air. 


XX.  [Workman; 
open  air. 
Same 
organism 
from 
plates. 


XXI.  J  Physician; 
outdoor. 


12/13 


12/14 


12/14 


1/20 


Morphology 


Capsules. 


Lanceolate 
cocci  in 
pairs  and 
short 
chains. 
Gram 
positive. 


Positive ; 
serum 
(Hiss). 


Inulin- 
Serum 
Water. 


3  days, 
clot. 


Lanceolate 
and 
round 
cocci  in 
pairs  and 
long 
chains. 
Gram 
positive. 


Lanceolate 
and 
round 
cocci  in 
pairs. 
Gram 
positive. 

Lanceolate, 
round,  and 
sometimes 
flattened 
cocci  in 
pairs  and 
chains. 
Gram 
positive. 

Lanceolate 
cocci  in 
pairs,  no 
chains. 
Gram 
positive. 


Positive ; 
serum 
(Hiss). 


Positive ; 
serum 
(Hiss). 


Positive; 
serum 
(Hiss). 


Positive ; 
serum 
(Hiss). 


48  hrs., 
clot. 


48  hrs., 
clot. 


24  hrs., 
clot. 


36  hrs. 
clot. 


Pathogenicity. 


4  c.c.  24-hr.  bouillon  culture 
subcutaneously  in  rabbit, 
""^-  1570  grams;  death  in  4 
days.  Extensive  sero-fibrin- 
ous  exudate  in  subcutaneous 
tissues;  small,  soft  spleen. 
Culture  from  heart  and  sub- 
cutaneous tissues  positive. 

Virulence  gradually  raised.  In 
various  rabbits  there  were 
produced  sero-fibrinous  and 
hemorrhagic  subcutaneous 
exudates,  subcutaneous  oede- 
ma, sero-purulent  peritonitis, 
pleuris3%  and  pericarditis, 
extensive  broncho-pneu- 

monia, and  haemorrhagic 
septiccemia.  After  passage 
through  16  rabbits,  0.00 1  c.c. 
24-hr.  bouillon  culture  kills 
rabbit,  wt.  480  grams,  in  12 
hours. 

4  c.c.  24-hr.  bouillon  culture 
subcutaneously  in  rabbit, 
wt.  2240  grams;  death  in  24 
hrs.  Fairly  extensive  sero- 
fibrinous exudate  in  sub- 
cutaneous tissues.  Spleen 
small  and  soft.  Cultures 
negative. 

One  24-hr.  blood-agar  culture 
subcutaneously  in  rabbit, 
vrt.  1240  grams;  death  in  7 
days.  Extensive  subcu- 
taneous congestion  and 
fibrinous  exudate ;  sero- 
purulent  peritonitis.  Cul- 
tures from  heart  and  perito- 
neum positive. 

One  24-hr.  blood-agar  slant 
intraperitoneally  in  rabbit, 
■^'t.  1380  grams.  Death  in 
35  days  from  secondary  in- 
fection. 


One  24-hr.  blood-agar  slant 
subcutaneously  in  rabbit, 
wt.  1700  grams;  death  in  36 
hrs.  No  local  lesions;  large, 
hard  spleen.  Cultures  and 
cover-slips  from  heart  posi- 
tive. 


After  passage  through  one 
rabbit,  i  c.c.  24-hr. bouillon 
culture  intraperitoneally  in 
rabbit,  wt.  890  grams;  death 
in  12  hrs.  No  local  lesions. 
Culture  from  heart  positive. 


40  Corajparative  Study  of  Pnewmococci  and  Streptococci 

TABLE  III  {Continued). 


No.  of 
Organism. 


XXII. 


XXIII. 


XXVII. 


XXVIII. 


XXXIII. 


XXXVIII.  Physician 


Source. 


Hospital 
interne. 


Laboratory 
janitor. 


Physician ; 
after  sea 
voyage. 


Laborer. 


Physician ; 
acute 
coryza ; 
nasal 
swab. 


Date. 


1/25 


1/28 


2/23 


2/27 


3/19 


4/6 


Morphology. 


Lanceolate 
cocci  in 
pairs. 
Gram 
positive. 


Lanceolate 
and  round 
cocci  in 
pairs  and 
short 
chains. 

Lanceolate 
and  round 
cocci  in 
pairs  and 
few  short 
chains. 
Gram 
positive. 

Lanceolate 
cocci  in 
pairs,  no 
chains. 
Gram 
positive. 


Lanceolate 
cocci  in 
pairs  and 
long 
chains 
Gram 
positive. 


Lanceolate 
and  round 
cocci  in 
pairs  and 
short 
chains. 
Gram 
positive. 


Capsules. 


Positive ; 
serum 
(Hiss). 


Positive ; 
serum 
(Hiss). 


Positive ; 
serum 
(Hiss). 


Positive ; 
serum 
(Hiss). 


Positive ; 
blood-agar 
(ordinary 
stains). 


Inulin- 
Serum 
Water. 


24  hrs. 
clot. 


24  hrs. 
clot. 


48  hrs., 
clot. 


48  hrs., 
clot. 


No  clot, 
TO  davs 


Positive ; 
serum 
(Hiss). 


Pathogenicity. 


One  24-hr.  blood-agar  slant 
subcutaneously  in  rabbit, 
wt.  680  grams;  death  in  36 
hrs.  Extensive  subcutane- 
ous fibrinous  exudate.  Small 
soft  spleen.  Cultures  from 
subcutaneous  tissues  and 
heart's  blood  positive. 

I  c.c.  24-hr.  bouillon  culture 
subcutaneously  in  rabbit, 
wt.  1200  grams;  death  in  11 
days.  Sloughing  sore  io 
abdominal  wall.  Cultures 
from  heart,  peritoneum,  and 
subcutaneous  tissues  nega- 
tive. 

One  24-hr.  blood-agar  slant 
subcutaneously  in  rabbit, 
wt.  1200  grams;  death  in  41 
days.      Secondary  infection, 


:  c.c.  saliva  in  rabbit,  wt.  390 
grams;  death  in  48  hrs.  Ex- 
tensive sero-fibrinous  exu- 
date in  subcutaneous  tissues. 
Cultures  from  heart  and  sub- 
cutaneous tissues  positive, 


48  hrs. 
clot. 


I 


One  24-hr.  blood-agar  slant 
subcutaneously  in  rabbit, 
wt.  900  grams;  death  in  4 
days.  Moderate  subcutane- 
ous fibrinous  exudate ;  spleen 
large  and  hard.  Cultures 
from  heart  positive,  i  c.c. 
24-hr.  bouillon  culture  sub- 
cutaneously in  rabbit,  wt. 
Qoo  grams ;  death  in  33  days. 
Much  emaciation.  Cultures 
from  heart  negative. 

After  passage  through  5  rab- 
bits, 0.5  c.c.  24-hr.  bouillon 
culture  intraperitoneally  in 
rabbit,  wt.  620  grams;  death 
in  12  hrs.  Sero-sanguineous 
peritonitis;  large,  hard 
spleen.  Cultures  from  peri- 
toneum and  heart  positive. 
Organism  produces  sero- 
fibrinous subcutaneous  exu- 
date, subcutaneous  oedema, 
and  fibrino-purulent  perito- 
nitis. 

After  passage  through  2  rab- 
bits, I  c.c.  24-hr.  bouillon 
culture  intraperitoneally  JD 
rabbit,  wt.  350  grams;  death 
in  5  days.  Extensive  fibrin- 
ous peritonitis;  spleen  en- 
larged and  soft.  Cultures 
from  peritoneum  and  heart 
positive.  In  other  animals 
subcutaneous  sero-fibrinous 
exudates  were  produced. 


Warfield  T.  Longcope  and  W.  W.  Fox  41 

In  this  group  have  been  included  all  the  organisms  which  re- 
sembled the  cocci  of  Series  B.  There  are  nineteen  which  could 
not  be  differentiated  by  the  methods  employed  from  the  or- 
ganisms classed  in  this  latter  group.  The  morphology  and  the 
staining  reactions  were  the  same;  capsules  could  always  be 
demonstrated  with  ease  by  Hiss'  method  and  the  cocci  stained 
by  Gram's  method.  On  blood-agar  the  same  watery  type  of 
growth  was  observed,  and  in  the  stock  serum  a  diffuse  cloud  and 
large  quantities  of  acid  were  produced.  Inulin  was  fermented 
and  inulin-serum  water  acidified  and  clotted  in  all  but  one  in- 
stance (A  XXXIII).  This  organism  is  comparable  to  B  X.  ■  It 
was  fairly  virulent  for  animals,  producing  extensive  fibrinous 
exudates  in  the  subcutaneous  tissues,  fibrino-purulent  peritonitis, 
and  the  hard,  swollen  spleen  infiltrated  with  fibrin. 

The  acid  production  in  inulin  was  sometimes  slower  and  less 
marked  than  with  the  B  series.  Indeed  in  the  whole  group  of 
organisms  which  fermented  inulin  quite  a  variation  existed  in 
the  amount  of  acid  produced  as  could  be  shown  by  tritration  with 
-^^  N.  sodium  hydroxide. 

The  organisms  belonging  to  Type  I  of  Series  A  were,  as  a  rule, 
much  less  virulent  for  rabbits  than  those  of  Series  B.  Many 
were  practically  non-pathogenic,  and  none  in  their  original  cul- 
tures showed  any  high  grade  of  virulence.  The  virulence  could, 
however,  be  exalted  by  successive  passages  through  animals. 
For  instance,  with  A  XVII,  after  passage  through  sixteen  rabbits, 
o.ooi  c.c.  of  a  twenty-four-hour  bouillon  culture  killed  medium- 
sized  rabbits.  The  elevation  of  virulence  was,  however,  in  some 
cases  very  difficult,  as  in  A  I,  where  it  required  passages  through 
nineteen  rabbits  to  produce  a  culture  capable  of  killing  rabbits  in 
doses  of  0.05  c.c.  of  a  twenty-four-hour  bouillon  culture.  The 
lesions  in  the  animals  which  died  were  in  every  respect  like  those 
produced  by  the  cocci  of  Series  B.  During  the  elevation  of 
virulence  a  certain  sequence  of  lesions  was  apt  to  occur.  First, 
extensive  fibrinous  exudates  in  the  subcutaneous  tissues  or 
peritoneum,  if  the  inoculation  was  made  intraperitoneally ;  later, 
fibrinous  or  fibrino-purulent  exudates  in  the  pleuree  or  pericar- 
dium, and  finally,  general  septicaemia  without  local  lesions.     Two 


42 


Comparative  Study  of  Pneumococci  and  Streptococci 


animals  lived  for  several  weeks  at  the  end  of  which  time  they 
died  of  a  general  septicaemia,  cultures  and  cover-slips  from  the 
heart's  blood  at  autopsy  showing  enormous  numbers  of  pneumo- 
cocci. 

The  organisms  of  Type  II  differed  in  many  respects  from  the 
above  description. 

TABLE  IV. 
Series  A.     Type  II. 


No.  of 

Inulin- 

Organism 

SoiATce. 

Date. 

Morphology. 

Capsules. 

Serum 
Water. 

Pathogenicity. 

IL 

Outdoor 

1 1/2 

Lanceolate 

Blood-agar ; 

No  clot, 

I  c.c.   24-hr.  bouillon  culture 

physician. 

and  round 
diplococci. 
Gram 
positive. 

suggestive 

10  days. 

subcutaneously     in     rabbit, 
wt.  345  grams.     Killed  acci- 
dentally after  5  days.     Cul- 
tures   from    blood    positive. 
No  subcutaneous  exudate. 

IV. 

Laboratory 

11/23 

Small 

Negative ; 

No  clot, 

I  c.c.  24-hr.  bouillon  culture 

assistant. 

round 
cocci  in 
pairs  and 
clusters. 
Gram 
positive. 

serum 
(Hiss). 

10  days. 

subcutaneously     in     mouse. 
Animal  lived. 
2  c.c.   24-hr.  bouillon  culture 
subcutaneously     in     rabbit, 
wt.  2000  grams.      Lived. 

VI. 

Hospital 

11/26 

Small  round 

Negative ; 

No  clot, 

2  c.c.  24-hr.  bouillon  culture 

resident. 

cocci  in 

pairs  and 

long 

chains. 

Gram 

positive. 

serum 
(Hiss). 

8  days. 

subcutaneously     in     rabbit, 
wt.  1400  grams;  lived.    One 
rabbit  during  immunization 
died  14  days  after  intraperi- 
toneal injection  of  3  c.c.  24- 
hr.   serum  bouillon  culture. 
General  septicaemia.        Cul- 
tures from  heart  and  peri- 
toneum positive.      No  other 
animals     could     be     killed, 
though  large  doses  were  used 
both  subcutaneously  and  in- 
traperitoneally. 

xin. 

Outdoor 

12/1 

Small, 

Negative ; 

No  clot. 

2  c.c.   24-hr.  bouillon  culture 

physician. 

round,  and 
flattened 
cocci  in 
pairs  and 
long 
chains. 
Gram 
positive. 
Long  chains 

serum 
(Hiss). 

6  days. 

intraperitoneally  in   mouse; 
death  in  6  days.     No  local 
lesions;    cultures  from  peri- 
toneum and  heart  positive. 
One  24-hr.  blood-agar  culttire 
intraperitoneally   in_  rabbit, 
wt.   1400  grams.      Lived. 

XXV. 

Gatekeeper. 

2/14 

Negative ; 

No  clot. 

One    24-hr.   blood-agar  slant 

1 

of  round 

serum 

10  days. 

subcutaneously     in     rabbit, 

1 

and  flat- 

(Hiss). 

wt.  1 190  grams;  death  in  14 

tened 

days.       Abscess    in    subcu- 

cocci. 

cutaneous  tissues.     Cultures 

Gram 

negative. 

positive. 

Warfield  T.  Long 

cope  and  W.  W.  Fox                     43 

TABLE   IV  (Continued). 

J.  of 

Inulin- 

J  inism. 

Source. 

Date. 

Morphology. 

Capsules. 

Serum 
Water. 

Pathogenicity. 

kxvi. 

Workman. 

2/17 

Small, 

Negative ; 

No  clot. 

One    24-hr.    blood-agar    slant 

f 

round,  and 

serum 

7  days. 

subcutaneously     in     rabbit, 

lanceolate 

(Hiss). 

wt.    650   grams;  death  in  3 

cocci,  often 

days.             Small     pea-sized 

in  pairs 

localized  abscess  at  point  of 

and  short 

inoculation.     Cultures  nega- 

chains. 

tive. 

Gram 

positive. 

XIX. 

Elevator 

3/14 

Small, 

Negative ; 

No  clot, 

One    24-hr.    blood-agar    slant 

I 

boy. 

round,  and 
lanceolate 
cocci  in 
pairs, 
groups, 
and 
chains. 
Gram 
positive. 
Lanceolate, 

serum 
(Hiss). 

8  days. 

subcutaneously  in  rabbit, 
wt.  680  grams;  death  in  41 
days.  No  local  lesions.  Cul- 
tures negative. 

'«iXX. 

Book- 

3/6 

Positive ; 

No  clot. 

One   24-hr.   blood-agar   slant, 

binder. 

round,  and 
oval  cocci 
in  pairs. 
Gram 
positive. 
Round  and 

serum 
(Hiss). 

8  days. 

subcutaneously  in  rabbit, 
wt.  430  grams;  death  in  27 
days.  No  local  lesions.  Cul- 
tures negative. 

xxv. 

Laborer. 

3/29 

Negative ; 

No  clot, 

One    24-hr.    blood-agar    slant 

lanceolate 

serum 

7  days. 

subcutaneously     in     rabbit, 

cocci  in 

(Hiss). 

wt.   909  grams;    death  in  7 

pairs  and 

days  from  rabbit  septicaemia. 

chains  and 

groups. 

Gram 

1 

positive. 

l^XVI. 

Pressroom 

3/29 

Round  and 

Negative ; 

No  clot, 

One    24-hr.    blood-agar    slant 

worker. 

lanceolate 
cocci  in 
pairs  and 
chains. 
Gram 
positive. 

serum 
(Hiss). 

7  days. 

subcutaneously  in  rabbit, 
wt.  950  grams;  death  in  14 
days.  Localized  abscess 
filled  with  white,  grumous 
material  at  point  of  inocu- 
lation. Cultures  from  ab- 
scess and  organs  negative. 

IXVII. 

Hospital 

4/1 

Lanceolate 

A  few 

No  clot. 

Three  24-hr.  blood-agar  slant 

resident. 

cocci  in 
pairs  and 
chains. 
Gram 
positive. 

organisms 
have 
capsules ; 
serum 
(Hiss). 

8  days. 

intraperitoneally  in  rabbit. 
Lived. 

XVIII. 

Physician. 

4/6 

Small, 

Negative ; 

No  clot. 

Three  24-hr.  blood-agar  slants 

round,  and 

serum 

8  days. 

intraperitoneally     in     small 

lanceolate 

(Hiss). 

rabbit.     Lived. 

cocci  in 

pairs  and 

chains. 

Gram 

positive. 

44  Comparative  Study  of  Pneuinococci  and  Streptococci 

TABLE  IV  {Continued). 


No.  of 

Inulin- 

Organism. 

Source. 

Date. 

Morphology. 

Capsules.  ■ 

Serura 
Water. 

Pathogenicity. 

XXXIX. 

Physician. 

4/6 

Small, 

Negative ; 

No  clot. 

One    24-hr.    blood-agar   slant 

round,  and 

serum 

9  days. 

subcutaneously     in     rabbit 

slightly 

(Hiss). 

wt.  340  grams;  death  in  i; 

lanceolate- 

days.     No  local  lesions.  Cul- 

shaped 

tures  negative. 

cocci  in 

■ 

pairs  and 

If 

long 

,.l 

chains. 

■ 

XL. 

Laboratory 

4/24 

Round  and 

Negative ; 

No  clot, 

Three  24-hr.  blood-agar  slams 

assistant, 

lanceolate 

serum 

8  days. 

intraperitoneally   in   rabbit, 

same  as 

cocci 

(Hiss). 

wt.  300  grams;  death  in  12 

A  I. 

singly,  in 
pairs,  and 
groups. 
Gram 
positive. 

hrs.  Fibrino-purulent  peri- 
tonitis; spleen  large  and 
soft.  Cultures  from  heart 
and  peritoneum  positive. 

XLII. 

Laboratory 

4/19 

Round  and 

Negative ; 

No  clot. 

Two  24-hr.  blood-agar  slants 

assistant. 

lanceolate 
cocci  in 

pairs  and 

short 

chains. 

Gram 

positive. 

serum 
(Hiss). 

1 1  days. 

intraperitoneally  in  small 
rabbit.  Living  12  days 
after. 

XLIII. 

Physician ; 

4/26 

Small, 

Negative ; 

No  clot, 

2  c.c.  saliva  m  white  mouse. 

same  as 

round,  and 

serum 

10  days. 

Lived. 

XVII  A. 

flattened 

(Hiss). 

Mouse 

cocci  in 

lived.  Cul- 

pairs and 

■ 

tures  in 

chains. 

1 

plates  from 

T 

saliva. 

i 

Of  these  there  were  sixteen,  and  though  not  conforming  in 
every  particular  to  the  class  of  cocci  grouped  in  Series  C,  they 
came  much  closer  to  them  than  to  those  in  Series  B. 

They  appeared  usually  as  diplococci,  often  having  a  lancet- 
shape,  but  with  a  tendency  to  form  round  and  flattened  forms 
arranged  in  chains.  They  stained  positively  by  Gram's  method. 
Capsules  could  not  be  demonstrated  except  in  rare  instances  and 
then  only  with  the  special  stains  of  Hiss  when  they  were  quite 
indefinite.  The  growth  on  blood-agar  was  dryish  and  formed 
a  fine  granular  film.  So  characteristic  was  this  appearance  that 
it  could  frequently  be  predicted  from  the  blood-agar  growth 
whether  the  organisms  belonged  to  Type  I  or  Type  II,  and  in 
two  instances  a  mixed  culture  was  detected  by  this  means.     No 


Warfield  T.  Longcope  and  W.  W.  Fox  45 

acid  and  no  clot  was  ever  produced  in  inulin-serum  water.  In 
serum  there  was  no  cloud,  but  the  growth  settled  to  the  bottom 
as  a  granular  sediment.  Traces  of  acid  were  occasionally  pro- 
duced, and  in  this  respect  the  organisms  differed  slightly  from 
those  belonging  to  Series  C. 

As  a  rule,  these  organisms  showed  a  very  slight  grade  of  viru- 
lence. In  large  doses  they  did  sometimes  kill  rabbits,  but  the 
organism  could  rarely  be  cultivated  from  the  organs  at  autopsy. 
The  lesions  consisted  of  small  subcutaneous  abscesses  and  ulcers 
of  the  skin.  None  of  the  lesions  described  for  Type  I  and  Series 
B  could  be  produced  by  any  of  the  organisms  of  this  group. 

From  the  saliva  of  two  different  individuals  organisms  be- 
longing to  both  Type  I  and  Type  II  were  isolated  and  separated 
satisfactorily.  In  both  instances  the  cocci  were  recovered  from 
the  tissues  of  a  mouse  inoculated  with  2  c.c.  of  saliva. 

By  treating  these  organisms  with  human  blood,  according  to 
the  method  of  Wright  and  Douglas  for  the  demonstration  of  the 
opsonic  power  of  the  serum,  it  was  discovered  that  the  leucocytes 
of  normal  individuals  ingested  the  cocci  of  Series  C  and  Series 
A,  Type  II,  as  far  as  they  were  tested,  but  did  not  take  up  the 
organisms  belonging  to  Series  B  and  Series  A,  Type  I.  Un- 
fortunately it  was  only  possible  to  make  tests  with  a  few  mem- 
bers of  each  group,  so  that  these  results  are  rather  suggestive 
than  conclusive.  By  reason  of  the  above  differences  it  was 
thought  justifiable  to  separate  this  large  group  of  cocci  of  Series 
A  into  the  two  types  just  described. 

Altogether  forty  specimens  of  saliva  were  examined.  The 
method  was  to  inoculate  2  c.c.  of  saliva  subcutaneously  into  a 
white  mouse,  and  at  autopsy  to  make  cultures  from  the  sub- 
cutaneous tissues,  peritoneum,  and  heart's  blood.  In  certain 
instances  plate  cultures  were  also  made  from  the  saliva.  In  four 
instances  we  succeeded  in  obtaining  the  same  organism  from 
plates  and  from  the  inoculated  animal. 

From  the  40  cases,  organisms  were  isolated  ^t,  times,  or  in 
82.5  %.  In  19  cases,  or  in  47.5  %,  Type  I  was  obtained;  in  16 
cases,  or  in  40  %,  Type  II.  In  two  instances  both  organisms 
were  isolated. 

In  the  table  below,  the  organisms  are  tabulated  according  to 
the  occupation  of  the  individual  from  whom  the  saliva  was  taken. 


46              Comparat 

iVe  Study  of  Pneumococci 
TABLE  V. 

a/ifZ  Strexjtococci 

Occupation. 

&> 

O  K-a 

6 
> 

> 

to 

2 

o 
p. 

0 

S 

0 

0. 

r 

c 

Hospital  interne 

Laboratory    assistants 
and  janitors 

Physicians     living      in 
tox\-n  and  country.   . 

Laborers  working  out- 
of-doors  

II 

9 

12 

7 
3 

8 
8 

ID 

6 

3 

3 

I 

2 

I 

72.7 
88.8 
83-3 
85-7 
100 

6 

5 
5 
3 

54-5 
55-5 
41.6 
42.8 

2 

3 

5 
3 
3 

18.2 

33-3 
41.6 
42.8 
100 

Laborers ;     indoor     oc- 
cupation   

42 

35 

7 

83.3  % 

19 

45-2  % 

16 

38.1% 

Though  the  number  of  cases  is  small,  still  the  relative  per- 
centages of  the  two  t3^pes  of  organisms  and  of  the  total  number 
of  positive  results  remain  fairly  constant. 

In  the  table  below  the  organisms  are  arranged  according 
to  the  month  of  the  3^ear  in  which  the  saliva  was  examined. 


TABLE  VI. 


,  ^ 

-^  > 

£-13 

'^^'^ 

Month. 

^■°c 

^A 

^ 

> 

H-; 

0 

S 

S 

"^^  s  s 

M 

c 

•_ 

0. 

u 

H 

^ 

2 

fi^O. 

C-i 

c 

H 

0, 

November 

9 

6 

3 

66.6 

3 

33-3 

3 

33-3 

December 

9 
3 
5 
7 
9 

9 
3 

0 
0 

100 
100 

8 
3 

88.8 
100 

I 
0 

II. 2 

Tanuarv .             

0 

Februarv 

4 
5 
8 

I 
2 

I 

80 

71.4 

88.8 

2 

I 
2 

40 

14.2 

22.2 

2 

4 
6 

40 

March 

57-2 

April    

66.6 

42 

35 

7 

83-3  % 

19 

45-2  % 

16 

38.1% 

Warfield  T.  Longcope  and  W.  "W.  Fox  47 

In  this  table  the  variation  in  the  percentages  of  the  two  types 
of  organism  is  very  great,  and  though  the  number  of  examinations 
is  too  small  to  permit  conclusions  to  be  drawn,  still  the  results 
are  suggestive. 

In  November,  the  percentage  of  the  true  pneumococcus  type 
is  not  very  large;  in  December  and  January  it  increases  enor- 
mously, to  fall  again  gradually  to  a  low  level  in  March  and  April. 
In  one  instance  (A  I,  Type  I) ,  a  fairly  virulent  member  of  Type 
I  was  recovered  from  the  saliva  in  November,  while  in  April 
(A  XL)  only  organisms  belonging  to  Type  II  were  obtained  from 
the  saliva  of  the  same  individual.  Again,  in  another  instance,  in 
December,  a  very  virulent  organism  (A  XVII,  Type  I)  was  re- 
covered from  the  saliva  both  by  plating  and  animal  inoculation, 
while  in  April  the  saliva  from  the  same  person  produced  no  effect 
in  mice,  and  in  plates  only  organisms  belonging  to  Type  II 
(A  XLIII)  could  be  obtained. 

These  experiments  suggest  that  the  pneumococcus  is  not  to 
be  found  constantly  in  the  mouths  of  40  %  or  50  %  of  healthy 
individuals,  as  one  might  at  first  suppose  from  the  tabulated 
results.  The  technique  which  we  used  was  the  same  throughout 
our  work,  so  that  the  failure  to  obtain  a  large  percentage  of 
pneumococci  in  the  spring  months  could  scarcely  depend  upon 
this  factor.  Of  course  the  series  of  cases  is  far  too  small  to 
enable  one  to  draw  conclusions,  but  the  results  suggest  that 
during  the  winter  months  the  pneumococcus  has  a  wide  distri- 
bution, and  that  at  this  time  a  large  percentage  of  healthy  in- 
dividuals harbor  virulent  pneumococci  in  their  buccal  cavity. 
These  months  precede  those  in  which  pneumonia  is  most  preva- 
lent. It  is  almost  certain  that  some  persons  always  have  virulent 
pneumococci  in  their  saliva. 

In  conclusion  we  should  like  to  express  our  thanks  to  Dr. 
Alfred  Stengel,  Dr.  C.  Y.  White,  and  Dr.  Nisbit,  who  placed 
the  Pepper  Clinical  Laboratory  at  our  disposal  for  the  animal 
investigations,  and  who  facilitated  this  part  of  the  work  by  many 
kindnesses. 


A  STUDY  OF  PNEUMOCOCCI  AND  ALLIED  ORGANISMS 
IN  HUMAN  MOUTHS  AND  LUNGS  AFTER  DEATH. 

By  CHARLES  NORRIS,  M.D.,  and  ALWIN  M.  PAPPENHEIMER,  M.D.« 
(Front  the  Laboratories  of  Bellevue  and  Allied  Hospitals,  New  York  City.) 

Our  studies  have  been  confined  especially  to  a  determination 
of  the  prevalence  of  the  pneumococcus  in  normal  lungs,  and  in 
lungs  which  presented  various  lesions.  At  the  same  time,  we 
have  tried  to  determine,  by  experimental  methods,  how  justly 
one  may  draw  inferences  with  regard  to  the  flora  of  the  living 
lung  from  cultural  findings  after  death. 

Ntimerous  investigations  have  shown  that  the  pneumococcus 
is  more  or  less  constantly  present  in  a  variety  of  pulmonary 
lesions,  especially  in  lobar  pneumonia.  The  pneumococcus  has 
been  isolated  from  a  considerable  proportion  of  apparently 
healthy  lungs,  both  human  and  animal,  that  have  been  examined 
after  death.  A  similar  micro-organism,  or  one  closely  related 
to  it,  has  been  found  in  the  mouths  of  many  healthy  human 
beings. 

The  question  of  the  presence  of  bacteria  in  normal  lungs  has 
long  been  an  urgent  one,  because  of  its  obvious  bearing  upon 
the  determination  of  the  occurrence  of  the  infectious  diseases 
of  this  organ.  If  organisms  are  present  in  aU  lungs,  it  may  be 
supposed  that  the  pneumococcus  will  be  more  frequently  found 
in  the  lungs  of  patients  exposed  for  some  time  to  a  hospital 
atmosphere.  This  point,  too,  we  have  tried  to  decide,  and  for 
this  reason  we  have  classified  our  cases  into  the  following  three 
groups,  viz: 

I.  Patients  dying  outside  of  hospital. 

II.  Patients  dying  in  hospital  within  twenty-four  hours  of 
admission. 

>  Aided  by  a  grant  from  the  Commission  for  the  Investigation  of  Acute  Re- 
spiratory Diseases  of  the  Department  of  Health  of  New  York  City. 

48 


Charles  Norris  and  Alwin  M.  Pappenheimer  49 

III.  Patients  dying  in  hospital  twenty-four  hoiirs  or  longer 
after  admission. 

This  report  embodies  the  results  of  the  bacterial  examination 
of  a  series  of  forty-two  (42)  human  lungs.  The  cases  were  taken 
at  random,  so  as  to  comprise  a  variety  of  lesions.  There  were 
included  cases  which  died  in  the  wards  of  Bellevue  Hospital, 
and  a  lesser  number  of  coroner's  cases,  which  died  either  outside 
the  hospital  or  in  the  hospital  within  twenty-four  hours  after 
admission. 

These  lungs  were  selected  from  subjects  examined  within 
twenty -four  hours  after  death.  The  bodies  were  removed  from 
the  wards,  usuaUy  within  an  hour  after  death,  and  kept  in  cold 
storage  until  examination. 

METHODS    EMPLOYED    IN    THE    STUDY    OF    THE    BACTERIA    IN    THE 
MOUTH   AND    LUNGS. 

Isolation. — The  isolation  of  the  organisms  studied  was  carried 
out  in  the  following  manner:  The  lung  to  be  cultivated  was 
clamped  before  removal,  by  a  large  hysterectomy  clamp,  which 
firmly  occluded  the  large  vessels  and  bronchus.  This  was  done 
to  prevent,  as  far  as  possible,  the  aspiration  of  fluid  or  mucus 
from  the  trachea,  during  the  manipiilation  of  removing  the  lungs. 
The  surface  of  the  lung  was  thoroughly  seared  with  a  large  knife 
over  the  posterior  portion  of  the  lower  lobe.  The  juice  was 
obtained  in  the  usual  manner,  by  stabbing  the  sterile  area  with 
a  heated  Nuttall  spear,  and  was  then  thinly  streaked  on  glycerine- 
agar  plates.  The  fluid  suspension  of  material  from  the  lungs 
for  the  inoculation  of  the  mice  was  obtained  as  follows:  With 
sterile  forceps  and  scissors,  a  large  piece  of  lung  was  excised  from 
the  seared  area  and  cut  into  small  pieces  in  a  sterile  Petri  dish ; 
in  cases  where  the  juice  expressed  was  insufficient  a  little  broth 
was  added.  From  0.5  to  i.o  c.c.  was  inoculated  into  the  skin 
of  the  back.  In  the  majority  of  cases  white  mice  were  used,  but, 
owing  to  the  difficulty  of  obtaining  a  sufficient  number,  we  were 
compelled  (in  a  few  instances  which  are  noted)  to  use  colored  or 
spotted  mice. 


60  Study  of  Pneumococci  and  Allied  Organisms 

From  the  lung  plates,  after  from  twenty -four  to  forty-eight 
hours,  sub-cultures  were  made  on  Loeffler's  blood-serum  from 
pneumococcus-  or  streptococcus-like  colonies ;  usually  as  many 
as  half  a  dozen  were  transplanted. 

From  the  heart's  blood  of  mice  dying  after  inoculation,  streak 
plates  were  made  on  gtycerine-agar,  and  the  colonies  which  had 
developed  after  from  twenty-four  to  forty-eight  hours  were  sub- 
cultured  on  Loeffler's  blood-serum.  In  case  the  colonies  varied 
in  appearance,  several  sub-cultures  were  always  made  from  each 
type. 

For  the  isolation  of  the  pneumococcus  from  saliva  we  have 
depended  almost  wholly  upon  the  subcutaneous  inoculation  of 
mice  with  a  small  amount  of  mucus  from  the  mouth.  This  was 
obtained  at  autopsy,  on  a  cotton  swab,  which  was  then  shaken 
in  broth  and  the  suspension  injected.  The  subsequent  pro- 
ceedings were  identical  with  those  above  described  for  the  lungs. 
Glycerine-agar  plates  were  made  in  a  few  instances,  but,  on 
account  of  the  difficulty  encountered  in  obtaining  satisfactory 
plates,  this  was  not  done  as  a  routine. 

Morphology. — The  morpholog}^  of  the  bacterial  flora  of  the 
lung  was  studied  in  cover-slip  preparations  and  in  sections  of 
lung  tissue.  As  a  routine  stain  for  the  demonstration  of  cap- 
sules, the  two  staining  methods  as  described  by  Hiss  ^  were 
employed;  in  smears  from  the  lung  tissue,  a  Gram  preparation 
was  also  made.  Sections  were  stained  for  bacteria  by  the  Gram- 
Weigert  method. 

Cover-slip  preparations  from  the  heart's  blood  of  mice,  from 
typical  colonies  on  plates,  from  the  primary  sub-cultures  on 
Loeffler's  medium,  were  examined  as  a  routine,  and  in  many 
instances  from  the  transplantations  on  different  media. 

Determination  of  Cultural  Characters. — For  the  study  of  cul- 
tural characteristics,  transplants  were  made  from  the  primary 
Loeffler  sub-ctdtures  upon  the  following  media:  broth,  gelatine, 
glycerine-agar  slants,  litmus-milk.  Hiss'  i  %  inulin-serum 
water  (1:3).  In  addition,  the  fermentative  activities  of  a  cer- 
tain number  of  what  we  may  for  the  present  call  the  different 
^Journal  of  Experimental  Medicine,  1905,  vi,  317. 


Charles  Korris  and  Alwin  M.  Pappenheimer  51 

strains  of  the  pneumococcus,  as  well  as  the  streptococcus,  were 
studied  on  a  series  of  sugar  media  containing  dextrose,  lactose, 
maltose,  saccharose,  mannit,  or  glycogen,  and  on  dextrin  and 
soluble  starch. 3 

Determination  of  Virulence. — This  was  limited  to  the  inocula- 
tion of  mice  with  half  or  whole  cultures,  grown  for  twenty-four 
hours  on  glycerine-agar  slants.  Unfortunately,  we  were  not  in 
a  position  to  determine  accurately  the  virulence  of  the  various 
strains,  owing  to  the  insufficient  supply  of  mice  at  our  disposal. 
Because  of  this  scarcity  we  rarely  were  able  to  ascertain  the 
virulence  of  the  organism  in  fresh  isolations. 

DESCRIPTION   AND   CLASSIFICATION   OF   THE   PNEUMOCOCCI,    STREP- 
TOCOCCI,  AND  ALLIED  FORMS,  FOUND  IN  THE  LUNGS  AND 

MOUTHS. 

In  consonance  with  the  opinions  of  more  recent  observers,  we 
believe  that  the  older  criteria  relied  upon  for  the  differentiation 
of  pneumococci  and  streptococci  are  insufficient.  Even  the 
fermentative  activities  upon  which  so  much  stress  has  recently 
been  laid  have  not  been,  in  our  hands,  an  infallible  guide,  espe- 
cially with  reference  to  the  identification  and  classification  of 
the  intermediate  types  of  these  two  closely  related  cocci,  of 
which  many  have  been  encountered. 

We  must  likewise  take  exception  to  the  older  and  perhaps 
still  widely  prevalent  view,  that  streptococci,  unlike  the  pneu- 
mococci, may  not  produce  a  bacterisemia  in  mice,  as  in  many 
cases  we  were  able  to  isolate  streptococci  in  cultures  from  the 
heart's  blood  of  mice  taken  immediately  after  death. 

In  order  to  describe  the  bacteria  we  have  encountered  during 
the  course  of  this  work  we  have  been  forced,  somewhat  against 
our  will,  to  adopt  an  arbitrary  classification  founded  mainly  upon 

3  The  sugars  were  obtained  from  Merck.  The  inulin  employed  by  us  in 
the  earlier  determinations  was  obtained  from  Merck.  This  inulin  was  found 
highly  unsatisfactory,  from  the  presence  of  resistant  spores.  All  otir  later 
work  was  done  with  Kiliani's  inulin,  "Eimer  and  Amend."  Many  of  our 
earlier  cultures  were  tested  again  upon  this  same  inulin.  Kahlbaum's  soluble 
starch  was  used  for  the  starch-serum  water,  in  the  strength  of  two  per  cent. 


52  Study  of  Pneumococci  and  Allied  Organisms 

the  variations  in  morphological  and  physiological  characters. 
The  most  important  and  constant  of  these  are  the  presence  or 
absence  of  capsules,  and  the  fermentation  or  non-fermentation 
of  inulin.     Our  grouping  of  these  cocci  is  as  follows : 

GROUP  I. 

Typical  pneumococci. — By  this,  we  designate  those  diplococci  which  ferment 
iniilin  with  acid  formation,  and  coagulation  of  the  serum;  which  possess 
readily  stained  capsules,  not  only  in  the  blood  of  mice,  but  on  various  media; 
which  give  a  diffuse  cloudiness  in  broth,  and  grow  as  fine,  colorless,  delicate, 
translucent  colonies  on  glycerine  agar,  and  which  are  virulent  to  mice.  We 
have  met  with  a  number  of  organisms  which,  although  fermenting  inulin  with 
slight  acid  production,  do  not  coagulate  the  serum.  This,  we  consider,  indi- 
cates, in  general,  feeble  growth. 

GROUP  II. 

Streptococcus  ntucosus. — By  this  term,  we  refer  to  a  series  of  organisms  which 
differ  from  the  typical  pneumococcus  in  their  abundant,  moist,  or  mucous 
growth  upon  various  solid  media  and  in  the  more  constant  production  of  chains 
upon  media,  but  which  resemble  the  pneumococcus  in  possessing  a  constantly 
demonstrable  capsule,  in  their  fermentative  activity,  and  in  their  virulence 
to  mice. 

In  conformity  with  the  observations  of  other  observers,  we  have  found  that 
the  capsules  of  these  diplococci  are  more  easily  demonstrable  than  those  of  the 
typical  pneumococcus,  and  that  there  are  certain  variations  in  the  structure 
of  the  capsules,  which  we  have  not  observed  in  those  of  the  pneumococcus. 
We  have  found  that  the  cocci  of  this  group  grow  more  constantly  and  abund- 
antly at  room  temperature  in  gelatin. 

We  have  not  as  yet  been  able  to  decide  definitely,  whether  the  group  con- 
stitutes a  distinct  variety  of  the  pneumococcus,  or  whether  the  mucous  char- 
acter of  the  growth  and  chain  formation  is  dependent  upon  variations  in  the 
media.  Thus  we  have  found  that  on  dry  Loeffler's  serum  tubes  the  growth  of 
these  organisms  may  be  dry,  whereas  on  wet  tubes  it  tends  to  be  mucous-like 
in  character.  We  have  also  seen  organisms  resembling  the  pneumococcus,  after 
passage  through  mice,  assume  a  viscid  character  of  growth.  In  fact,  there 
seem  to  be  numerous  gradations  between  these  two  types  in  this  respect. 

GROUP  III. 

Diplococci  resembling  pneumococci  which  possess  no  demonstrable  capsule. — 
No  capsules  have  been  demonstrated  in  these  diplococci,  either  in  the  heart- 
blood  of  mice,  or  on  culture  media  after  repeated  examination.  They  produce 
acid  in  inulin,  but  do  not  coagulate  the  serum.  In  all  other  respects,  they 
resemble  pneumococci. 


Charles  Norris  and  Alwin  M.   Pappenheimer  53 

GROUP  IV. 

Diplococci  resembling  streptococci  which  possess  no  demonstrable  capsule. — No 
capsules  have  been  demonstrated  in  these  diplococci,  either  in  the  heart-blood 
of  mice,  or  on  culture  media.  They  are  active  inulin  fermenters.  These  cocci, 
although  fermenting  inulin  with  acid  production,  have  greater  points  of  simi- 
larity to  the  streptococcus  than  to  the  pneumococcus.  They  grow  abundantly 
in  artificial  media,  produce  clouding,  or  fiocculi  and  granules  in  broth,  grow 
readily  at  room  temperature,  and  show  a  marked  tendency  to  chain  formation. 

GROUP  V. 

Typical  streptococci. — These  have  no  capsules,  do  not  ferment  inulin  with 
acid  production,  and  grow  readily  at  room  temperature.  They  produce 
clouding  in  broth,  or  form  granules  or  flocculi  adherent  to  the  sides  of  the  tubes. 

SOURCES    OF    ERROR    IN   TECHNIQUE. 

It  is  obvious  that  there  are  many  possibilities  of  error  in  the 
isolation  of  pneumococcus-like  organisms  from  the  lungs  by 
methods  above  detailed.  On  account  of  the  similarity  in  the 
colonies  on  glycerine-agar,  and  even  on  ascitic-agar,  there  is 
always  difficulty  in  distinguishing  on  inspection  pneumococcus 
from  short-chained  streptococcus  colonies,  and  in  consequence 
the  pneumococcus,  though  present  in  small  numbers,  may 
escape  detection,  for  it  is  obviously  not  practical  to  sub-culture 
more  than  a  limited  member  of  suspicious  colonies. 

Overgrown  plates  are  another  but  not  a  frequent  source  of 
trouble.  The  organisms  responsible  for  this  are  not  only  those 
which  are  introduced  through  natural  errors  in  technique,  but 
also  those  which  we  have  found  more  or  less  constantly  present 
in  the  lungs  cultivated  after  death,  as  verified  by  a  thorough 
examination  of  cover-slip  preparations  from  the  lung  tissue. 

Again,  another  source  of  error  lies  in  the  natural  insuscepti- 
bility of  white  mice,  and  even  more  so  of  certain  strains  of 
colored  mice,  to  infection  by  pneumococci.  Thus  it  has  been 
found  by  us  that  colored  mice  are  more  resistant  to  infection 
than  white  mice  when  inoculated  with  similar  doses.  Moreover, 
it  has  been  found  that  in  some  of  our  cases  even  a  white  mouse 
does  not  succumb  to  the  inoculation  of  a  comparatively  large 
amount  of  expressed  lung  juice,  although  cover-slip  preparations 


54  Study  of  Pneumococci  and  Allied  Organisms 

of  the  lungs  have  shown  typical  capsulated  diplococci,  positive 
to  Gram.  For  this  reason,  so  far  as  it  has  been  possible,  we  have 
inoculated  two  mice  from  each  case. 

Still  another  though  less  frequent  and  serious  difficulty  is 
that  encountered  in  the  isolation  of  pneumococci  in  streak  agar 
plates  from  the  heart  blood  of  mice  dying  of  mixed  infection. 
In  a  certain  number  of  cases  we  have  found  Bacillus  mucosus 
capsulatus  to  be  the  predominating  organism;  in  other  cases, 
non-capsulated  cocci  which  we  have  not  as  yet  identified.  In 
a  certain  number  of  cases  also,  though  pneumococcus-like  organ- 
isms were  present  in  smears  from  the  lung,  the  mice  inoculated 
died  from  streptococcus,  Bacillus  mucosus  capsulatus,  or  other 
infections.  On  the  other  hand,  we  have  encountered  capsulated 
cocci  in  the  heart's  blood  of  mice,  as  well  as  upon  our  lung 
plates,  which  failed  to  grow  on  transplantation  upon  glycerine- 
agar  or  Loeffier's  blood-serum. 

With  this  brief  introduction,  we  can  now  present  more  readily 
the  result  of  our  work  largely  in  tabulated  form.  The  tables 
which  follow  must  be  briefly  described. 

The  first  column  of  the  tables,  headed  "Case  Number,"  indi- 
cates the  number  of  the  case  in  our  series.  The  next  column, 
the  "  Accession  Nimiber,"  refers  to  the  entry  number  of  each  case 
in  the  autopsy  accession  book  of  the  hospital.  The  Roman 
numerals  I,  II,  III,  IV,  and  V  are  those  we  have  used  to  designate 
the  five  groups  into  which  we  have  divided  the  pneumococci, 
streptococci,  and  the  cocci  allied  to  them.^  The  addition  sign 
(  +)  or  the  zero  sign  (o)  opposite  each  case  indicates  that  a  coccus 
of  this  group  has  or  has  not  been  isolated. 

4  I. — Pneumococcus. 
II. — Streptococcus  mucosus. 
III. — Diplococci  without  capsules,  which  ferment  inulin  only  with  acid 

production. 
IV. — Diplococci  without  capsules,  which  are  active  inulin  fermenters,  and 

which  closely  resemble  streptococci. 
V. — Streptococci. 


Charles  Norris  and  Alwin  M.  Pappenheimer 


55 


PNEUMOCOCCI,    STREPTOCOCCI,    AND    ALLIED    MICRO-ORGANISMS    IN 
MOUTHS    AFTER    DEATH. 

In  fourteen  cases  the  mouths  were  cultivated  by  the  methods 
sufficiently  described  above.  We  append  the  analysis  of  our 
findings : 

TABLE  I. 

ANALYSIS     OF     PNEUMOCOCCI,     STREPTOCOCCI,     AND     ALLIED     MICRO-ORGANISMS, 
ISOLATED  FROM  THE   MOUTH  AFTER  DEATH. 


Case 
No. 

Accession 
No. 

Group 

Group 
■      II. 

Group 
III. 

Group 
IV. 

Group 
V. 

Unidenti- 
fied. 

I 
2 
3 
4 
5 
6 

7 
8 

9 
10 
II 
12 
13 
14 

82 

98 

105 

no 

153 

164 

165 

166 

167 

170 
172 
191 
199 
233 

+ 
0 
0 
+ 
0 
0 
+ 
+ 
0 
0 
0 
+ 
0 
0 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 

0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
0 
+ 
0 

0 
0 
0 
0 
+ 
0 
0 
0 
0 
0 
0 
0 
0 
0 

0 
+ 
+ 
0 
0 
+ 
0 
0 
0 
+ 
0 
+ 
0 
0 

(?) 

+ 
+ 

Total      14  cases 

5 

0 

I 

I 

5 

2 

The  pneumococcus,  as  shown  by  Table  I,  was  isolated  five 
(5)  times,  the  streptococcus  five  (5)  times,  and  intermediate 
organisms  twice.  Of  these  twelve  cases,  all  but  three  were  in 
the  hospital  over  twenty-four  hours.  Two  died  with  lobar 
pneumonia;  in  one  of  these,  the  mouse  injected  with  mouth 
secretion  died  of  streptococcus  infection,  and  this  organism,  in 
pure  culture,  produced  a  bactericemia  fatal  within  twenty-four 
hours  in  a  mouse  inoculated  with  three-fourths  of  a  glycerine- 
agar  growth  twenty-four  hours  old. 

The  virulence  of  the  cocci  isolated  was  tested  in  eight  (8) 
cases.  Three  (3)  of  these  were  pneumococci,  four  (4)  were 
streptococci,  and  one  (i)  Group  IV.  One  of  the  streptococcus 
cultures  proved  to  be  non-virulent.     All  the  others  killed  the 


56  Study  of  Pneumococci  and  Allied  Organisms 

mice  in  from  eighteen  to  forty-eight  hours,  partial  or  whole  cul- 
tures being  inoculated. 

All  the  mice  inoculated  with  mouth  secretion  died,  in  most 
cases,  within  twenty-four  hours.  Mice  inoculated  with  the 
secretion  from  two  cases  died  with  infections  other  than  pneu- 
mococcus  or  streptococcus.  From  a  third  case,  one  of  the  mice 
inoculated  died  after  four  (4)  days,  no  organisms  being  recovered 
in  smears  or  by  culture ;  the  second  mouse  of  the  same  case  re- 
mained alive. 

These  few  observations  seem  to  show  that  the  streptococci 
isolated  from  the  mouth  were  quite  as  virulent  as  the  pneumo- 
cocci. The  pneumococci  obtained  from  two  (2)  cases  not  dying 
of  lobar  pneumonia  were  found  to  be  quite  as  virulent  as  those 
obtained  from  pneumonic  lungs  post-mortem.  Further  con- 
clusions, however,  as  to  the  general  virulence  of  the  pneumo- 
cocci obtained  from  the  mouth  do  not  seem  to  be  justified. 

We  have  made  no  attempts  to  determine  the  frequency  of 
pneumococci  in  the  mouths  of  ward  patients  or  attendants  on 
account  of  an  insufficient  supply  of  mice. 

PNEUMOCOCCI,     STREPTOCOCCI,    AND    ALLIED     BACTERIA    ISOLATED 
FROM    THE    LUNGS    AFTER   DEATH. 

Forty- two  (42)  cases  were  examined.  The  analysis  of  our 
findings  follows  in  tabulated  form  (Table  II). 

Table  III  shows  the  number  of  cases  in  which  the  various 
groups  of  cocci  have  been  found,  either  alone  or  in  association 
with  the  other  groups. 

We  are  now  in  a  position  to  analyze  the  facts  presented  above, 
in  Table  IV,  which  requires  a  brief  explanation.  The  cases  cul- 
tivated in  this  research  have  been  divided  into  three  classes, 
according  to  whether  the  lungs  were  normal  in  the  gross  or  pre- 
sented the  lesions  of  lobar  pneumonia,  or  were  the  seat  of  a 
variety  of  other  lesions.  Each  of  these  classes  has  again  been 
divided  into  three  classes,  viz.,  those  cases  dying  outside  the 
hospital,  those  dying  in  the  hospital  within  twenty-four  hours  of 
admission,  and  those  dying  twenty-four  hours  or  more  after 
admission. 


Charles  Norris  and  Alwin  M.  Pappenheimer 
TABLE  II. 


57 


ANALYSIS  OF  THE  DIPLOCOCCI  ISOLATED  FROM  THE  LUNGS. 5 


Cas 

e        Accession 

Group 

Group 

Group 

Group 

Group 

Unidenti- 

No 

No. 

I. 

II. 

III. 

IV. 

V. 

fied. 

I 

43 

0 

0 

0 

0 

0 

2 

45 

0 

0 

0 

0 

+ 

3 

46 

0 

0 

0 

0 

+ 

4 

49 

+  {^y 

0 

0 

+ 

+ 

5 

52 

0 

0 

0 

+ 

0 

6 

54 

0 

0 

0 

+ 

0 

7 

63 

+ 

0 

+ 

+ 

0 

8 

68 

+  (?) 

0 

0 

0 

0 

9 

70 

+  (?) 

0 

0 

0 

0 

lO 

71 

0 

+ 

0 

0 

0 

II 

82 

+ 

0 

+ 

0 

0 

12 

98 

0 

0 

0 

0 

+ 

13 

102 

0 

0 

0 

0 

+ 

14 

105 

+  (?) 

0 

0 

0 

+ 

15 

no 

+ 

0 

0 

0 

0 

16 

113 

0 

0 

0 

0 

+ 

17 

114 

+  (?) 

0 

0 

0 

+ 

18 

123 

0 

0 

0 

0 

0 

+ 

19 

140 

+ 

+ 

+ 

0 

0 

20 

147 

0 

0 

0 

0 

0 

21 

149 

0 

0 

0 

+ 

0 

22 

153 

0 

0 

+ 

0 

0 

23 

160 

0 

+ 

0 

0 

0 

24 

164 

0 

0 

0 

0 

+ 

25 

165 

+ 

0 

0 

0 

+ 

26 

166 

+ 

0 

0 

0 

+ 

27 

167      . 

0 

0 

0 

0 

+ 

28 

170 

+ 

0 

0 

0 

+ 

29 

172 

+ 

0 

0 

0 

0 

30 

175 

0 

0 

0 

0 

+ 

31 

191 

+ 

0 

0 

0 

0 

32 

199 

0 

0 

+ 

0 

+ 

33 

C  I 

+ 

0 

0 

+ 

0 

34 

CII 

0 

0 

0 

+ 

+ 

35 

C  III 

+ 

0 

0 

0 

0 

36 

C  IV 

+ 

0 

0 

0 

0 

37 

C  V 

0 

0 

0 

0 

+ 

38 

C  VI 

0 

0 

0 

0 

+ 

39 

C  VII 

+ 

0 

0 

0 

0 

40 

233 

+ 

0 

+ 

0 

+ 

41 

237 

+ 

0 

0 

0 

0 

42 

258 

+ 

0 

0 

0 

0 

'  See  explanation  of  Table  I,  p.  456. 

« The  cocci  of  Group  I  which  are  marked  ( ?)  were  not  absolutely  identified 
as  pneumococci,  but  they  in  all  probability  belong  to  the  group,  and  they  have 
therefore  been  included. 


58 


Study  of  Pneumococci  and  Allied  Organisms 
TABLE  III. 

ANALYSIS  OF  THE  DIPLOCOCCI  ISOLATED  FROM  LUNGS. 


Group  of 
Organisms.' 

No.  of 
Times  Found. 

Accession  No.  of  Cases. 

I  alone, 

lO 

68,  70,  no,  172,  191,  C  III,  C  IV,  C  VII,  237, 

258. 

II       " 

2 

71,  160. 

III       " 

I 

153- 

IV       " 

3 

52,  54,  149. 

V       " 

lO 

45,  46,  98,  102,  113,  164,  167,  175,  C  V,  C  VI. 

I  +  II 

o 

I  +  III 

I 

82. 

I  +  IV 

I 

CI. 

I  +  V 

5 

105,  114,  165,  166,  170. 

II  +  III 

o 

II  +  IV 

o 

III  +  V 

o 

III  +  V 

199. 

IV  +  V 

CII. 

I  +  III  +  IV 

63- 

11+  II  +  III 

140, 

I]+  IV  +  V 

lU  III  +  V 

49. 

233- 

Unidentified. 

2 

123,  147- 

The  data  presented  in  Table  IV  may  be  thus  briefly  reviewed : 

I. NORMAL    LUNGS. 

a.  Two  cases  dying  outside  of  hospital. — In  one,  the  pneumococcus  was 
isolated;  in  the  other,  only  a  streptococcus. 

h.  Four  cases  dying  within  twenty-four  hours  of  admission. — In  these  (63,  68, 
153,  223)  the  pneumococcus  was  isolated  three  times;  one  of  these  cultures 
(68),  however,  may  be  considered  questionable. 

It  is  interesting  to  note  that  likewise  in  three  of  the  cases  organisms  of 
Group  III  (non-capsulated  inulin  fermenter)  were  found,  and  in  two  of  the 
cases  streptococci  (Group  V). 

c.  Eight  cases,  dying  twenty-four  hours  or  more  after  admission. — The  pneu- 
mococcus was  obtained  in  two  cases:  in  166,  where  it  was  associated  with 
streptococcus  (Group  V);  and  in  258,  where  it  occurred  alone.  In  two  cases 
(71  and  160),  Streptococcus  mucosus  (Group  II);  in  one  case  (149),  Group  IV; 
in  four  cases  (98,  166,  167,  C  V.),  streptococci  (Group  V). 

The  pneumococcus,  if  68  be  called  a  true  pneumococcus,  was  thus  isolated 
five  times.  It  is,  perhaps,  noteworthy,  that  pneumococcus  was  isolated  from 
the  cases  dying  outside,  and  those  less  than  twenty-four  hours  in  hospital,  in 
four  out  of  six  cases,  66  %,  whereas  it  was  isolated  in  only  two  out  of  eight 
cases  which  had  been  in  the  hospital  over  twenty-four  hours,  24  %. 

'  See  explanation  of  Table  I. 


Charles  Norris  and  Alwin  M.  Pappenheimer 


59 


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60  Study  of  Pneumococci  and  Allied  Organisms 

II. LOBAR    PNEUMONIA. 

Thirteen  (13)  cases,  one  dying  outside  the  hospital,  three  (3)  within  twenty- 
four  hours  of  admission,  and  nine  (9)  after  a  residence  in  hospital  of  twenty- 
four  hours  or  over.  The  pneumococcus  was  isolated  from  seven  of  these  cases; 
this  includes  one  non-inulin  fermenter  (114),  which,  however,  exhibited  a 
capsule  after  the  second  passage  through  mouse,  the  culture  having  been  taken 
from  the  pneumonic  lobe. 

Cultures  were  made  in  seven  of  the  thirteen  cases  from  the  consolidated 
lobe;  in  two,  from  the  non-consolidated.  Of  the  remaining  four  (4)  cases, 
which  showed  organization  of  the  exudate  (so-called  organizing  pneumonia), 
cultures  were  made  from  the  involved  lobe  three  (3)  times;  from  the  unin- 
volved  lobe,  once. 

From  the  seven  lobes  showing  a  typical  pneumonia  with  exudate,  in  three 
cases  (140,  C  VII,  237)  typical  pneumococcus  (Group  I)  was  obtained;  once 
(140)  associated  with  Streptococcus  mucosus  (Group  II);  in  two  cases 
(105,  the  organism  growing  poorly,  and  114,  above  mentioned)  two  typical 
pneumococci  were  isolated,  associated  with  streptococci  (Group  V) ;  in  another 
case  (199)  Group  III,  and  also  a  streptococcus,  Group  V;  in  another  (113) 
Group  V  was  isolated,  no  other  organisms  being  found  in  this  case. 

Of  two  cases  cultivated  from  the  uninvolved  lobes,  one  (no)  gave  a  typical 
pneumococcus;  the  other  (147)  showed  B.  mucosus  capsulatus  in  pure  cultiire 
from  the  lungs  in  plates,  and  from  the  mouse  in  plates,  no  pneumococci  having 
been  found  in  the  cover-slip  preparations  made  from  the  heart's  blood  of  mouse. 

In  three  cases  where  the  lobes  with  organizing  pneumonia  were  cultured, 
in  one  (43)  no  bacteria  were  found  in  plates,  the  single  mouse  inoculated 
remaining  alive;  in  two  cases  (46  and  102)  streptococcus  (Group  V)  alone  was 
isolated. 

In  one  case  of  organizing  pneumonia  (165),  in  which  the  involved  lobe  was 
cultured,  a  pneumococcus  (Group  I)  and  a  streptococcus  (Group  V)  were 
isolated. 

III. LESIONS    OTHER   THAN    LOBAR    PNEUMONIA. 

Of  the  fifteen  remaining  cases,  none  died  outside  the  hospital;  two  were  in 
hospital  less  than  twenty-four  hours;  in  one  of  these  (C  I)  pneumococcus 
and  an  organism  of  Group  IV  were  found;  in  the  other  (C  III)  pneumococcus 
alone  was  found. 

Of  the  thirteen  cases  over  twenty-four  hours  in  hospital,  the  pneumococcus 
was  found  in  four  cases;  in  two  cases  (49  and  70)  organisms,  probably  pneu- 
mococci (Group  I),  were  obtained.  In  Case  49,  organisms  of  Groups  IV  and  V 
were  also  isolated;  in  70,  no  other  organisms  were  found.  In  one  case  (123) 
an  unidentified  non-capsulated  diplococcus,  which  did  not  grow  on  trans- 
plantation from  the  lung  plates,  was  obtained;  the  mouse  inoculated  with  lung 
tissue  remained  alive. 

From  the  tabiilated  summary  of  Table  IV  we  see  that  the  total 
number  of  cases  cultured  was  forty- two,  and  that  in  all  but  two 


Charles  Norris  and  Alwia  M.  Pappenheimer  61 

(2)  cases  micro-organisms  were  obtained.  From  these  cases, 
pneumococci  and  streptococci  were  obtained  in  practically 
similar  percentages — that  is,  in  50  %.  These  results,  as  far  as 
the  pneumococcus  is  concerned,  agree  with  those  obtained  by 
Beco,  50  %  pneumococci  and  45  %  streptococci  in  human  lungs. ^ 
Boni,^  in  lungs  of  pigs,  has  also  obtained  similar  percentages. 
Durck,^o  in  pneumonic  limgs  of  children,  obtained  pneumococcus 
in  cases  of  lobar  pneumonia,  some  of  which  were  organizing  or 
unresolved  pneumonias,  in  seven  out  of  thirteen,  52  %,  strepto- 
cocci being  found  in  the  same  proportion. 

Discussion  of  the  Significance  of  the  Data  above  Summarized. — 
A  consideration  of  these  facts  suggests  a  number  of  interesting 
questions :  Are  the  pneumococci  present  in  the  exudate  of  lobar 
pneumonia  similar  to  the  pneumococci  which  have  been  found 
in  the  mouths  of  normal  persons,  presumably  as  harmless  in- 
habitants? If  so,  they  reach  the  lungs  either  through  the 
lymphatics  or  the  vascular  channels  at  some  time  during  Hfe, 
or  by  inhalation,  lodging  there  as  harmless  saprophytes  until 
some  as  yet  unknown  change  in  their  host  causes  them  to  acquire 
increased  virulence.  A  second  possibility  is  that  pneumococci 
of  greater  virulence  than  those  found  in  the  mouth  are  inhaled 
with  the  air  current  during  life,  determining  the  onset  of  the 
disease.  However,  it  is  still  an  open  question  whether  pneumo- 
cocci are  present  in  the  lungs  of  normal  persons  during  life. 
May  not  their  presence  in  normal  lungs,  as  found  by  post-mortem 
examinations,  be  explained  by  a  terminal  septicaemia,  such  as 
has  been  established  for  the  streptococcus  and  other  micro^ 
organisms,  or  do  the  pneumococci  of  the  mouth  gain  access  to 
the  air  vesicles  with  the  saliva,  either  aspirated  during  the  death 
agony  or,  later,  by  gravitation  consequent  upon  the  manipula- 
tion of  the  body  after  death,  in  its  transportation  from  the 
wards  to  the  morgue? 

These  problems  complicate  the  interpretation  of  the  various 
factors  concerned  in  the  production  of  lobar  pneumonia.     With 

8  Beco,  Archiv.  d.  la  medecine  experimentale,  1889,  xi,  317. 

9  Boni,  Deutsch.  Arch.  f.  klin.  Med.,  1901,  Bd.  69. 
loDurck,  Deutsch.  Arch.  f.  klin.  Med.,  1897,  Bd.  58. 


62  Study  of  Pneumococci  and  Allied  Organisms 

the  more  theoretical  aspects  of  the  question,  such  as  the  dis- 
turbance in  balance  between  the  host  and  the  inciting  agent  of 
the  disease,  we  have  not  attempted  to  cope,  except  in  so  far 
as  we  have  roughly  determined  the  virulence  for  mice  of  the 
organisms  isolated  in  our  mixed  series  of  cases. 

The  statistics  obtained  by  the  investigators  mentioned  above 
and  by  oiirselves  might  lead  one  to  the  inference  that  bacteria 
of  the  pneumococcus  or  streptococcus  group  exist  during  life  in 
practically  all  lungs,  whether  normal  or  diseased.  But,  as  we 
have  already  suggested,  grave  theoretical  objections  may  be 
urged  against  such  a  conclusion.  Though  we  have  been  able  to 
obtain  these  organisms  in  97  %  +  of  our  cases,  the  possibility, 
as  suggested  above,  that  the  pneumococci  so  frequently  present 
in  the  mouth  may  reach  the  lungs  by  aspiration  during  the 
death  agony,  or  after  death,  during  the  transportation  of  the 
bodies  from  the  wards  to  the  morgue,  is  no  fanciful  one.^^ 

The  mouth  and  nose  are  frequently  found  at  autopsy  filled 
with  fluid  admixed  with  frothy  and  bloody  fluid,  or  vomitus. 
It  is  quite  evident,  under  such  conditions,  that,  by  mere  force  of 
gravity  and  the  fluid  communication  existing  between  the  upper 
and  lower  air-passages,  micro-organisms  may  find  their  way  from 

1 »  Because  of  its  possible  bearing  upon  the  gravitation  of  fluid  from  the  buccal 
cavity  to  the  lungs,  it  seems  worth  while  to  describe  in  detail  the  various 
manipulations  to  which  the  body  is  subjected  during  the  interval  between 
death  and  post-mortem  examination.  After  being  formally  pronounced  dead 
by  a  member  of  the  house  staff,  the  body  is  rolled  in  a  shroud,  the  jaw  sup- 
ported by  a  four-tailed  bandage,  and  cotton  plugged  into  the  mouth  and  other 
orifices.  Within  an  hour  or  less,  the  cadaver  is  ready  for  transportation  to  the 
morgue.  It  is  lifted  by  the  shoulders  and  heels,  to  a  four-wheeled  truck,  which 
has  been  rolled  to  the  bedside.  During  this  manoeuvre,  with  the  sagging  of  the 
body  the  head  is  of  necessity  at  a  higher  level  than  the  thorax.  From  the 
ward,  the  body  is  wheeled  directly  to  the  morgue, — a  distance  of  about  150 
yards.  Then  again  it  is  lifted  from  the  truck  to  a  wooden  frame,  which  is 
placed  upon  the  floor  next  to  the  carriage,  and  lying  upon  this,  it  is  raised,  feet 
foremost,  to  a  compartment  in  the  cold-storage  chamber.  Here  it  remains 
until  removed  to  the  autopsy  room.  In  this  final  handling  of  the  body,  the 
wooden  tray  is  slid  from  the  compartment,  the  head  lowest,  and  the  body  is 
caiYied  upon  this  wooden  frame  to  the  adjacent  room.  The  body  is  then  rolled 
upon  its  side,  abruptly  jolted,  and  the  head  raised  and  lowered  several  times. 
The  entire  proceeding,  therefore,  involves  considerable  disturbance  of  the 
body  before  the  necessary  examinations  are  made. 


Charles  Norris  and  Alwin  M.  Pappenheimer  63 

the  mouth  to  the  small  air  spaces ;  or,  again,  by  the  compression 
of  the  chest  wall  in  moving  the  body  a  false  respiratory  excur- 
sion may  take  place,  causing  the  replacement  of  the  air  in  the 
lungs  by  fluid  from  the  mouth  and  trachea.  If  this  takes  place, 
it  then  seriously  vitiates  the  value  of  inferences  drawn  from 
cultural  findings  after  death,  when  applied  to  the  living  lung. 

In  order  to  determine  the  frequency  of  this  occurrence  by  ex- 
perimental methods,  we  have  had  recourse  to  rabbits,  which  have 
been  given  small  doses  of  broth  cultures  of  B.  prodigiosus  just 
before  or  after  death.  Furthermore,  we  have  introduced  broth 
cultures  of  B.  prodigiosus  (a  half  drachm  or  less)  into  the  mouths 
of  patients  who  had  died  in  the  wards,  but  before  removal  of  the 
bodies  to  the  morgue. 

The  method  which  we  have  used  for  the  isolation  of  B.  pro- 
digiosus in  the  mouths  and  lungs  of  rabbits  and  cadavers  was  the 
following :  the  mouth  was  scraped  with  a  sterile  cotton  swab ;  a 
suspension  was  then  made  in  melted  agar  tubes,  and  plates  with 
one  dilution  poured.  The  mouth  plates  were  made  in  every 
case  in  order  to  make  sure  that  the  patients  had  received  the 
culture,  and  thus  they  served  as  control  for  the  lung  plates. 
From  the  lung  a  considerable  amount  of  expressed  juice,  ob- 
tained by  squeezing  and  crushing  a  piece  of  excised  lung,  was 
distributed  in  melted  agar  tubes  and  pour-plates  made.  Both 
series  of  plates  were  then  kept  at  room  temperature  and  ob- 
served until  the  appearance  of  typical  red  colonies,  or  for  several 
weeks.  Abundant  growths  of  various  organisms  were  regularly 
obtained  in  these  plates.     In  no  case  have  we  seen  that  the 

development  of  numerous  colonies  has  inhibited  the  pigment 
production.  ^2 

In  nineteen  (19)  cases,  half -drachm  or  smaller  portions  of 
broth  cultures  of  B.  prodigiosus  were  introduced  into  the  mouth 
by  the  interne  after  having  pronounced  the  patients  dead. 
Characteristic  red  colonies  of  B.  prodigiosus  developed  on  the 

»2  A  few  control  experiments  were  made,  to  test  this  point;  tubes  were 
inoculated  with  varying  proportions  of  dysenteric  faeces,  and  broth  cultures  of 
B  prodigiosus.  These  showed  that  pigment  formation  by  this  culture  of  B. 
prodigiosus  was  not  inhibited  by  the  excessive  growth  of  other  bacteria. 


64  Study  of  Pneumococci  and  Allied  Organisms 

pour-plates  from  the  mouths  of  these  nineteen  cases,  and  in  ten 
cases  more  or  less  numerous  colonies  of  B,  prodigiosus  developed 
in  the  lung  plates.  In  other  words,  in  a  little  over  50  %  of  the 
cases  evidence  was  obtained  that  micro-organisms  introduced 
into  the  mouths  of  patients  after  death  enter  the  Ixuigs.  In  half 
of  the  cases,  as  seen  from  Table  V,  numerous  colonies  developed 
on  the  Iting  plates. 

Since  it  was  impossible  to  personally  control  the  giving  of  the 
cultures  after  death,  we  have  not  included  in  the  table  nine  cases 
in  which  the  mouth  plates  were  negative.  We,  however,  believe 
that  of  these  nine  cases  some  received  the  cultures,  but  that  the 
B.  prodigiosus  failed  to  develop  its  characteristic  color  upon  the 
mouth  plates.  We  have  positive  knowledge  that  this  occurred 
in  one  case  (279,  not  included  in  table),  the  cotton  swab  of  the 
mouth  being  stained  pink.  Moreover,  as  above  stated,  our  B. 
prodigiosus  developed  pigment  in  over-crowded  plates ;  neverthe- 
less, as  is  well  known,  pigment  formation  is  subject  to  a  number 
of  disturbing  factors  which  are  not  readily  controlled  or  perhaps 
even  known. 

In  support  of  this  view  it  may  be  stated  that  in  one  case,  175, 
abundant  colonies  developed  in  the  lung  plates,  whereas  none 
appeared  in  the  mouth  plates ;  this  case  has  not  been  included 
in  the  final  estimate  of  the  percentages  of  positive  results.  For 
these  reasons,  we  believe  that  the  percentages  given  are  too  low 
for  the  mouth  as  well  as  for  the  Iting  plates. 

It  is  certainly  a  striking  observation  that  likewise  in  about 
fifty  per  cent.  (50  %)  of  our  forty-one  cases  the  pneumococcus 
was  isolated.  It  seems  a  fair  inference  from  this  coincidence 
that  the  utmost  caution  must  be  observed  in  the  interpretation 
of  the  cultural  findings  of  the  lung  from  post-mortem  statistics. ^^ 

13  In  five  (5)  cases,  the  prodigiosus  culture  was  received  shortly  before  the 
exitus  lethalis.  In  one  case  (82,  alcoholic  delirium),  numerous  colonies  de- 
veloped in  both  the  mouth  and  the  lung  plates.  In  two  cases  (98,  105),  the 
mouth  plates  alone  developed  colonies,  the  lung  plates  being  negative.  In  case 
1 10,  negative  results  were  obtained  in  the  mouth  and  lung  plates ;  this,  however, 
is  readily  accounted  for  by  the  fact  that  the  culture  was  administered  twenty- 
four  and,  again,  fourteen  hours  before  exitus.  Another  of  the  negative  cases 
(CV),  where  no  colonies  developed  on  the  lung  plates,  is  readily  enough  ac- 


Charles  Norris  and  Alwin  M.  Pappenheimer  65 

We  append  below  the  statistics  in  tabulated  form : 

TABLE  V. 

TABLE    SHOWING   THE    CASES    IN   WHICH    B.   PRODIGIOSUS,   INTRODUCED    INTO    THE 
MOUTH    POST-MORTEM,    WAS    RECOVERED    FROM    THE    MOUTHS    AND  LUNGS. 


No. 

Mouth. 

Lung. 

No. 

Mouth. 

Lung. 

i66 

+ 

214 

+  +  + 

+ 

i68 

+ 

+  +  + 

224 

+ 

— 

171 

+  +  + 

— 

233 

+  +  + 

+ 

172 

+ 

+ 

241 

+  +  + 

— 

181 

+  +  + 

+ 

258 

+  +  + 

— 

188 

+  +  + 

+  + 

262 

+  +  + 

+ 

199 

+ 

— 

278 

+  +  + 

— 

201 

+  +  + 

+  +  + 

281 

+  + 

+ 

205 

+ 

— 

288 

+  +  + 

+  +  + 

206 

+ 

Total  number  of  cases 19 

Mouth  positive 19 

Lung  positive 7 

Oiir  work  upon  the  entrance  of  B.  prodigiosus  from  the  mouths 
of  rabbits  to  their  lungs  has  been  unfortunately  confined  to  two 
(2)  rabbits.  One  rabbit  was  given  0.5  c.c.  of  a  broth  culture  of 
B .  prodigiosus  when  under  ether.  The  rabbit  died  twenty  minutes 
later  without  gasping.  The  body  was  then  slanted  upon  a  tray 
with  elevated  head.  (Post-mortem,  twenty-nine  hours  later.) 
No  colonies  of  B.  prodigiosus  developed  in  either  mouth  or  lung 
plates. 

The  other  rabbit,  dying  from  an  intraperitoneal  inoculation 
with  the  meningococcus,  was  given  0.5  c.c.  of  a  broth  culture, 
and  died  slowly  several  hours  later.  At  autopsy,  performed  at 
once  aiier  death,  pour-plates  were  made  from  the  mouth  and 
lungs.     Numerous  colonies  developed  on  the  mouth  plates ;  the 

counted  for  by  the  closure  of  the  glottis  from  oedema,  after  a  cut -throat  wound 
of  the  neck.  Thus,  colonies  of  prodigiosus  were  obtained  from  the  lungs  in  one 
out  of  three  cases,  33  %,  if  the  above  reasons  are  valid  for  excluding  the  two 
cases  (no  andCV).  As  regards  these  experiments,  however,  only  negative 
results  are  of  value,  since  one  cannot  make  sure  that  the  organisms  introduced 
during  life  did  not  reach  the  lung  after  death. 


66  Study  of  Pnemnococci  and  Allied   Organisms 

lung-plates  failed  to  show  red  colonies.  We  abandoned  this  line 
of  experimentation  for  the  reason  that  the  conditions  so  ob- 
viously differ  from  those  of  the  human  respiratory  tract,  which 
almost  constantly  contains  more  or  less  fluid,  at  least  after  death, 

THE  AGGLUTINATING   REACTIONS   OF  THE   PNEUMOCOCCUS,    STREP- 
TOCOCCUS,   AND    ALLIED    GROUPS    OF   DIPLOCOCCI. 

The  impracticabilty  of  using  agglutinating  reactions,  especially 
difficult  in  this  group  of  cocci,  led  us  to  forego  this  aid  to  species 
identification.  This  method,  as  has  been  repeatedly  shown, 
when  used  upon  species  belonging  to  the  same  group,  has  no 
differential  value,  unless  very  tedious  and  exact  quantitative 
serum  tests  are  employed.  For  these  reasons,  and  because  of 
the  impossibility  of  obtaining  sera  of  high  valency  within  the 
limited  time  at  our  disposal,  we  could  not  avail  ourselves  of  this 
method  during  the  greater  part  of  this  investigation. 

Before  giving  our  conclusions,  we  deem  it  best  to  give  the  re- 
sults that  we  obtained  upon  the  agglutinability  of  cultures  of 
species  belonging  to  the  various  groups  into  which  we  have  divided 
the  various  diplococci  that  we  encountered  during  the  progress 
of  our  work. 

We  were  able,  however,  at  the  end  of  our  work,  to  make  use 
of  the  method  of  obtaining  mass  cultures  for  agglutination  tests 
described  by  Prof.  Hiss,^"*  which,  in  his  hands,  has  yielded  such 
brilliant  results.  With  a  rabbit  immune  serum  giving  a  com- 
plete agglutination  at  i :  400  with  the  homologous  pneumococcus, 
which  we  obtained  through  the  kindness  of  Prof.  Hiss  and  Dr. 
Borden,  the  following  tests  were  made  the  day  after  receipt  of 
the  serum,  which  was  from  a  recent  bleeding : 

Broth  cultures  were  made  of  species  of  each  of  our  five  (5)  groups  of  cocci, 
strict  attention  being  paid  to  the  details  as  given  in  the  method  described  by 
Hiss,  in  the  article  above  referred  to.  The  results  of  oxxv  agglutination  tests 
obtained  with  the  pneumococcus  rabbit  serum,  mentioned  above,  and  with  a 
normal  rabbit  serum,  may  thus  be  briefly  described.  Two  diplococci  of  Group  I 
were  used;  the  first,  230,  a  typical  pneumococcus,  was  obtained  from  a  case  of 
suppurative  sphenoiditis  at  autopsy.     This  pneumococcus  did  not  lend  itself 

>*  Journal  of  Experimental  Medicine,  1905,  vii,  223. 


Charles  Norris  and  Alwin  M.  Pappenheimer  67 

to  the  agglutination  test,  at  least  with  the  mass  culttire  we  employed,  since 
the  control  settled  out  promptly,  and  clumps  were  speedily  developed.  The 
second  diplococcus,  C  VII,  another  typical  pneumococcus,  was  not  clumped  by 
the  normal  rabbit  serum,  after  twenty-four  hours,  the  control  showing  only  a 
few  small,  microscopic  clumps.  With  the  pneumococcus  serum,  large  fiocculi 
developed  in  the  i :  lo  dilution,  but  the  reaction  did  not  become  complete,  even 
after  twenty-four  hours,  the  fluid  still  being  cloudy.  Small  fiocculi  were,  how- 
ever, found  in  the  i :  200,  as  well  as  in  the  i :  1600  dilution,  unlike  the  control. 

Only  one  diplococcus  of  Group  II  was  tried,  namely  71,  a  typical  Streptococcus 
mucosus,  which  gave  a  slight  reaction  only  in  the  lowest  dilution,  namely,  i :  10. 
Normal  rabbit  serum  gave  no  reaction  in  a  similar  dilution. 

One  diplococcus  of  Group  III  was  tried  (199).  It  gave  with  the  pneumococcus 
rabbit  serum,  a  positive  reaction  at  i:  100,  but  no  complete  reaction  was  ob- 
tained even  at  1:10,  the  fluid  becoming  only  slightly  clearer  with  small  clumps 
on  the  sides  of  the  tubes.  The  reaction  in  the  i:  10  and  the  i:  100  dilutions 
differed  only  in  the  size  of  the  clumps.  Normal  rabbit  serum  had  no  action 
upon  this  diplococcus. 

Three  organisms  of  Group  IV  were  tried.  Culture  49  a  gave  a  slight  reaction 
with  pneumococcus  serum,  in  the  i:  10  dilution,  the  clumps  being  larger  than 
those  of  the  control.  After  twenty-fotir  hotirs,  all  the  dilutions  and  the  con- 
trol, however,  became  practically  clear.  Culture  149  was  not  affected  by  the 
pneumococcus  or  the  normal  rabbit  serum  in  any  dilution.  With  culture  C  II 
the  dilutions  and  the  control  were  practically  alike,  although  possibly  there 
were  larger  granules  in  the  lowest  dilution  than  in  the  control. 

Two  organisms  of  Group  V,  typical  streptococci,  were  tested.  Culture 
214  S  did  not  lend  itself  to  the  agglutination  test  on  account  of  the  prompt 
development  of  large  granules,  and  the  rapid  clearing  of  the  fluid  in  the  tubes 
in  the  control,  as  well  as  in  dilutions.  With  the  other  streptococcus,  153, 
larger  granules  were  found,  and  a  quicker  settling  in  the  lower  dilutions  occurred 
than  in  the  control,  which,  however,  after  twenty-fovir  hours,  became  perfectly 
clear. 

Thus,  if  one  may  judge  from  a  single  examination,  the  method 
of  mass  cultures  or  agglutination  tests  have  shown  that  only  in 
the  case  of  the  pneumococcus,  C  VII,  positive  agglutinations 
were  obtained,  and  even  with  this  organism  no  complete  re- 
action was  given  in  the  lowest  dilution,  namely,  i :  10,  after 
twenty-four  hours,  with  the  possible  exception  of  the  diplo- 
coccus 199,  of  Group  III. 

It  seems  to  us  somewhat  forced  to  draw  any  conclusions,  from 
the  single  agglutination  test  described  above,  upon  the  method 
of  using  mass  cultures,  but  we  believe  that  the  test  shows  that 
the  diplococci  which  we  have  not  identified  as  pneumococci — 
in  other  words,  all  those  belonging  to  other  groups  than  Group 


68  Study  of  Pneumococci  and  Allied  Organisms 

I — have  no  or  very  slight  agglutinating  affinities  to  the  pneiimo- 
coccus. 

Before  concluding,  it  may  be  well  to  state  that  we  fully  recog- 
nize the  arbitrariness  of  our  method  of  classifying  the  diplo- 
cocci  belonging  to  the  pneumococcus  and  streptococcus  groups. 

The  experience  we  have  gained  in  the  examination  of  a  large 
number  of  cultures  convinces  us  that  it  is  not  justifiable  to 
consider  all  diplococci  which  ferment  inulin,  pneumococci. 
Thus,  C  II  and  149  and  49a  are  rapid  inulin  fermenters,  but  in 
all  other  respects  they  resemble  streptococci.  It  is  of  interest 
to'note  in  connection  with  49a,  that  its  property  of  fermenting 
inulin  is  a  most  variable  one.  Thus,  on  passage  through  the 
first  mouse,  after  its  primary  isolation  from  the  lungs  upon 
glycerine-agar,  it  lost  its  fermenting  action  upon  inulin,  only  to 
regain  it  on  further  cultivation  upon  serum  water  made  from 
the  same  stock  of  inulin.  Further  passages  through  mice  did 
not  affect  this  function.  Again,  later  cultures  of  49  a  lost  their 
capacity  to  ferment  inulin,  but  regained  it  on  transplantation, 
and  are  now  active  and  rapid  fermenters.  Cultures  C  II  and 
149  have  never  changed,  having  always  been  active  fermenters. 

We  consider  that  the  presence  of  capsules  like  the  inulin  re- 
action can  not  be  regarded  as  an  infallible  guide  for  the  differ- 
entiation of  these  diplococci,  especially  when  the  Hiss  capsule 
methods  are  employed,  for  the  reason  that  we  have  encotmtered 
streptococci  with  distinct  capsules. 

The  cocci  of  what  may  be  called  the  intermediate  groups,  III 
and  IV,  it  may  be  of  interest  to  state,  have  been  foimd  in  the 
blood  during  life,  and  have  been  recovered  from  the  pial  exudate 
of  cases  of  meningitis.  A  further  study  of  these  interesting 
diplococci  is  greatly  needed. 

CONCLUSIONS. 

The  following  conclusions  may  be  drawn,  based  upon  the  re- 
sult of  our  researches : 

I .  Organisms  of  the  pneumococcus  or  streptococcus  group  are 
present  in  the  lungs  of  practically  all  cases,  whether  normal  or 


Charles  Norris  and  Alwin  M.  Pappenheimer  69 

showing  a  variety  of  lesions ;  strictly  speaking,  they  were  found 
by  us  in  forty  out  of  forty-two  cases,  or  in  95  %  of  our  series. 

2.  The  pneumococci  and  the  streptococci  were  obtained  in 
practically  similar  percentages — that  is,  in  50  %  of  the  cases. 

3.  Pneumococci  were  not  obtained  more  frequently  in  the  small 
series  of  patients  exposed  for  some  time  to  hospital  atmosphere ; 
our  tables  show  the  contrary  to  obtain.  The  number  of  cases 
examined  were,  however,  insufficient,  and  the  findings  may  thus 
be  accidental,  and  hence  of  no  value. 

4.  Test  micro-organisms,  namely,  small  portions — half  a 
drachm  or  less — of  B.  prodigiosus,  introduced  into  the  human 
mouth  after  death,  were  conveyed  to  and  recovered  from  the 
lungs  by  culture  in  a  little  over  half  of  the  cases  in  which  this 
experiment  was  tried.  The  test  micro-organisms  are,  we  be- 
lieve, conveyed  to  the  lungs  with  the  fluid  which  collects  in 
mouths  of  persons  after  death,  and  which  in  many  cases  collects 
just  before  death.  The  numerous  manipulations  entailed  in  the 
removal  of  the  body  from  the  wards  to  the  morgue  greatly 
facilitate  the  entrance  of  any  fluid  from  the  pharynx  and  buccal 
cavity  into  the  lungs. 

It  follows  logically,  from  the  results  obtained  in  this  experi- 
ment, that  the  cultural  findings  after  death  are  no  guide  to  the 
bacterial  contents  of  the  lungs  during  life,  and  that  any  deductions 
made  from  such  findings  are  unreliable  and  deceptive.  Granted 
that  our  explanation  be  correct,  there  is  every  reason  to  believe 
that  any  of  the  micro-organisms  present  in  the  mouths  and 
pharynx  and  in  many  cases  in  the  stomach  contents  may  enter 
the  lungs  and,  if  the  conditions  be  suitable,  increase  in  numbers, 
during  the  time  between  death  and  the  examination  of  the  lungs. 

5.  There  exists,  perhaps,  more  frequently  than  has  hitherto 
been  suspected,  a  series  of  diplococci,  intermediate  between  the 
typical  pneumococci  and  streptococci.  The  diplococci  of  this 
type  have  been  found  in  forty  (40)  per  cent,  of  our  cases. 

The  differential  diagnosis  of  these  atypical  diplococci  from 
the  pneumococci  and  streptococci  is  a  difficult  one,  depending,  as 
it  does,  upon  general  cultural  characteristics.  No  single  char- 
acter, such  as  the  presence  of  capsules  or  the  fermentation  of 


70  Study  of  Pneumococci  and  Allied  Organisms 

inulin,  virulence,  etc.,  has  been  found  to  be  a  certain  criterion. 
The  few  agglutinative  reactions  we  have  made  seem  to  show 
that  these  intermediate  diplococci,  those  of  Groups  II,  III,  and 
IV,  have  no  or  only  slight  agglutinative  affinities  to  the  typical 
pneumococcus.  Further  tests  must,  however,  be  made  with  the 
various  methods  at  our  disposal  before  this  statement  can  be 
accepted  as  final. 

These  diplococci  are  of  interest  from  the  fact  that  they  have 
been  found  in  the  blood  during  life,  and  in  the  pial  exudate  of 
cases  of  meningitis,  endocarditis,  etc. 

6.  Our  studies  have  thrown  no  light  whatever  upon  the  con- 
ditions which  determine  the  onset  of  lobar  pneumonia  in  ap- 
parently healthy  persons.  Moreover,  we  have  been  unable  to 
draw  conclusions  as  to  the  presence  of  pneumococci  in  the  lungs 
during  life,  or  as  to  the  channels  by  which  they  gain  access  thereto. 

In  concluding,  we  gladly  avail  ourselves  of  the  opportunity  of 
acknowledging  our  indebtedness  to  Prof.  T.  Mitchell  Prudden 
for  help  and  advice  freely  given  in  the  course  of  this  work,  and 
to  the  various  members  of  the  house  staff  of  Bellevue  Hospital 
for  their  aid  and  assistance  in  the  pursuit  of  these  researches, 
especially  to  Dr.  Frank  Erdwurm. 


STUDIES  ON  THE  PNEUMOCOCCUS. 

By  CHARLES  W.  DUVAL,  M.D.,  and  PAUL  A.  LEWIS,  M.D. 
{From  the  Pathological  Laboratory  of  the  Boston  City  Hospital.) 
Plate  XXIX. 

The  work  on  the  pneumococcus  at  the  Boston  City  Hospital, 
under  the  auspices  of  the  Commission  for  the  Investigation  of 
x\cute  Respiratory  Diseases,  of  the  Department  of  Health  of  the 
City  of  New  York,  was  begun  November  i,  1904,  and  continued 
until  July  i,  1905.  The  director  of  the  hospital  laboratory,  Dr. 
F.  B.  Mallory,  encouraged  us  to  accept  the  proposals  of  the  Com- 
mission, placed  the  facilities  of  the  laboratory  at  our  disposal, 
and  has  maintained  a  constant  interest  in  the  progress  of  the 
investigation.  The  physicians  of  the  hospital's  clinical  ser- 
vices, both  visiting  staff  and  house  physicians,  have  given  us 
every  assistance  in  procuring  material.  On  the  part  of  the  Com- 
mission we  have  had  the  support  and  advice  of  Dr.  Theobald 
Smith.  The  photomicrographs  we  present  were  taken  for  us  by 
Dr.  S.  B.  Wolbach.  To  all  of  these  gentlemen  we  extend  thanks 
for  their  interest  in  the  work  and  their  invaluable  assistance. 

The  purpose  of  the  investigation  was  to  ascertain,  so  far  as 
possible  with  the  material  under  our  control,  the  distribution  of 
the  pneumococcus,  and  by  a  subsequent  comparative  study  of 
cultures  isolated  to  see  if  there  might  be  a  constant  variation  in 
any  character  or  set  of  characters  coincident  with  the  source  of 
origin.  We  wish  to  report  as  concisely  as  possible  the  data  which 
bear  directly  on  these  questions. 

One  of  the  constant  difficulties  which  has  confronted  us 
throughout  the  work  has  been  the  separation  of  the  pneumo- 
coccus from  Streptococcus  pyogenes.  The  inulin  test  of  Hiss 
having  been  proposed  as  an  absolute  method  of  differentiating 
these  two  species,  we  give  in  detail  our  experience  with  it  in  the 
form  in  which  it  was  originally  proposed  and  in  the  modification 
we  find  most  satisfactory. 

7i 


72  Studies  on  the  Pneumococcus 

In  the  course  of  the  work  we  have  twice  encountered  a  diplo- 
coccus  which  has  proved  identical  in  general  characters  with 
those  isolated  by  Howard  and  Perkins/  and  by  Richardson  2  in 
this  country  and  by  several  observers  abroad.  It  has  usually 
been  described  under  the  name  Streptococcus  capsulatus  or 
mucosus.  As  we  have  been  able  to  add  some  new  facts  to  those 
previously  reported,  we  give  our  experience  with  it  in  some  detail. 

Except  for  the  method  of  using  the  inulin,  our  technique  is  a 
combination  of  methods  previously  in  use  and  well  known  to 
investigators  who  have  studied  this  group  of  bacteria.  We 
therefore  make  the  briefest  possible  statement  of  it. 

METHODS. 

In  cultivating  the  pneumococcus  we  have  found  that  attention  to  the  com- 
position and  reaction  of  the  culture  medium  is  essential.  All  media  having 
bouillon  as  a  base  shovild  be  made  from  beef.  The  reaction  should  be  adjusted 
with  but  one  change,  if  possible,  to  between  0.4  and  0.8  %  acid  to  phenol- 
phthalein  (cold  titration).  Dextrose,  or  some  other  carbohydrate  fermentable 
by  the  particular  culture  in  question  must  be  present  in  quantity  of  from  0.5  to 

1  %,  or  growth  will  be  neither  constant  nor  abundant.     The  addition  of  from 

2  to  5  %  of  "fresh"  defibrinated  blood  to  dextrose  agar-agar  we  have  fovmd  to 
make  the  most  satisfactory  medium  for  routine  cultivation.  If  these  con- 
ditions are  fulfilled,  the  temperature  has  only  its  usual  influence  on  rapidity  of 
growth.  Growth  always  takes  place  at  room  temperature.  The  gelatin-agar 
mixtures  with  the  addition  of  dextrose  give  good  growth.  The  other  culture 
media  which  we  have  found  particularly  useful  for  cultivation  and  identifica- 
tion of  the  organism  are  litmus-milk  and  the  serum-water-sugar  mixtures  made 
according  to  the  formulse  of  Hiss.  The  lactose-serum  water  is  particularly 
useful.  It  is  coagulated  by  both  the  pneumococcus  and  the  streptococcus  as 
a  rule,  but  by  the  former  in  twenty-four  hours,  and  by  the  latter  only  after 
many  days.     It  cannot  be  considered  to  be  an  absolutely  differential  medium. 

In  making  isolations  we  have  placed  our  dependence  on  the  plating  method 
with  surface  seeding.  Dextrose  agar-agar  plates  are  poured  and  solidified. 
Several  drops  of  sterile  defibrinated  blood  are  scattered  over  the  surface  with 
a  finely  drawn  pipette.  The  particular  kind  of  blood  used  seems  to  be  unim- 
portant. With  a  sterile  platinum  loop,  several  drops  of  a  previously  made 
bouillon  or  salt-solution  suspension  of  the  material  to  be  plated  are  added  to  one 
of  the  drops  of  blood  on  the  plate.  After  mixing  thoroughly  with  the  loop,  a 
drop  or  two  are  carried  to  a  second  drop  of  blood,  and  after  mixing  again,  to  a 
third.  After  these  dilutions  are  accomplished,  the  drops  are  spread  as  evenly 
as  possible  over  the  plate.  If  the  agar-agar  is  firm  (1.7  to  2  %),  and  the  wire 
loop  soft,  this  can  be  accomplished  without  roughening  the  surface  of  the 

»  Jour,  of  Med.  Research,  1901,  vi,  163. 

»  Jour,  of  the  Boston  Soc.  of  the  Med.  Sciences,  v,  499. 


Charles  W.  Duval  and  Paul  A.  Lewis  73 

medium.  Colonies  to  be  studied  are  transferred  from  the  plates  to  the  dex- 
trose blood-agar.  The  isolation  from  the  heart's  blood  of  an  animal  after  sub- 
cutaneous or  intraperitoneal  inoculation  of  the  material  to  be  examined  is  less 
satisfactory  as  a  routine  method,  in  our  experience.  If  used  with  discretion,  the 
method  does  not  fail,  but  it  is  more  tedious  and  expensive.  We  will  say  more 
of  the  method  in  discussing  the  virulence  of  the  pneumococcus. 

The  identification  of  the  pneumococcus  cannot  be  made  by  any  one  feature 
or  test.  It  is  best  made  within  a  few  days  after  isolation  of  a  culture.  The 
capsule  is  then  more  certain  to  be  large  and  distinct,  and  the  lanceolate  diplo- 
coccus  form  is  more  marked.  As  the  period  of  cultivation  grows  longer,  the 
capsules  are  less  constantly  demonstrated.  The  cocci  become  smaller  and  the 
pairs  more  flattened.  Under  certain  conditions  which  we  do  not  fully  under- 
stand, but  which  seem  to  be  generally  unfavorable  to  active  growth,  the  most 
typical  pneumococcus  culture  may  grow  as  pairs  of  oval  and  flattened  cocci 
in  long  chains.  Many  individual  cocci  in  such  a  chain  will  appear  distinctly 
biscuit-shaped.  Figure  4,  however,  is  intended  to  demonstrate  that  very  long 
chains  do  occur  even  under  favorable  conditions  with  freshly  isolated  pneumo- 
coccus cultures.  In  such  cases  the  size  of  the  capsule,  the  larger  size  of  the 
individual  coccus,  and  the  shape  of  the  pairs  must  be  depended  on  for  the 
diagnosis. 

The  colony  on  blood-agar  varies  under  conditions  we  have  not  been  able  to 
control,  from  an  almost  invisible  dry,  fiat  colony,  or  a  raised,  moist,  or  dewdrop- 
like  gro-w^h,  to  a  dead  white,  viscid  colony  2  ram.  in  diameter,  resembling  that 
of  Staphylococcus  albus.  Not  much  dependence  can  be  placed  on  such  a  vari- 
able character  in  identifying  a  culture. 

Culturally  the  rapid  fermentation  of  lactose  in  serum  water,  the  rapid  coagu- 
lation of  litmus  milk,  and  the  fermentation  of  inulin  are  the  points  on  which 
most  dependence  must  be  placed,  although  any  one  of  these  characters  may  be 
absent  with  a  given  culture.  If  inulin  be  fermented  by  a  micro-organism  having 
in  general  the  characters  of  the  pneumococcus,  the  identity  is  established,  in  our 
experience.  That  there  are,  however,  non-inulin-fermenting  pneumococci,  we 
feel  certain.     Such  cultures  will  be  described  when  we  discuss  the  inulin  test. 

OCCURRENCE    AND    DISTRIBUTION  OF  THE  PNEUMOCOCCUS. 

In  the  study  of  the  distribution  of  the  pneumococcus,  material 
was  studied  from: 

I.  Autopsies  in  the  hospital. 

II.  Surgical  material  sent  to  the  laboratory. 

III.  Cerebro-spinal  fluid  obtained  by  lumbar  puncture. 

IV.  Sputum  of — 

A. — Pneumonia  cases. 

B. — Other  cases  with  bronchial  or  pulmonary  trouble. 

C. — Saliva  from  persons  in  good  health  at  work  in  the  hospital. 

I.  Autopsies. — The  efforts  of  this  laboratory  have  been  chiefly 
directed  to  the  study  of  autopsy  material.     We  will  report  the 


74  Studies  on  the  Pneumococcus 

results  of  the  bacteriological  examination  of  the  lungs  of  thirty- 
three  persons  dying  with  acute  lobar  pneumonia.  We  include 
also  the  bacteriological  report  of  examination  of  the  lung  in  four 
cases  in  which  death  was  ascribed  to  lesions  outside  the  lung, 
although  the  lungs  showed  areas  of  broncho-pneumonia,  and  in 
two  cases  in  which  there  was  no  evidence  of  pulmonary  disease. 

In  order  that  the  value  of  the  results  may  be  correctly  es- 
timated, some  explanation  is  necessary.  Early  in  the  course  of 
the  work  our  only  aim  was  the  isolation  of  the  pneumococcus. 
Thus  all  of  the  examinations  reported  bear  on  the  question  of  the 
general  distribution  of  the  pneumococcus.  All  of  the  cases  in 
which  the  pneumococcus  was  the  only  bacterium  isolated  from 
the  lung  belong  to  this  period  when  we  were  attempting  nothing 
but  its  isolation.  No  consideration  should  be  given  to  the 
negative  aspect  of  these  cases. 

When  the  local  distribution  of  the  pneumococcus  in  the  lung 
is  considered,  only  two  of  our  cases  are  of  importance.  These 
are  reported  in  detail  (Autopsy  1905,  47,  and  Autopsy  1905,  82). 

The  reported  data  which  have  reference  to  the  association  of 
bacteria  in  the  pneumonic  lung  are  not  complete.  Other  micro- 
organisms were  found  which  were  only  roughly  classified  and 
noted.  Especial  effort  was  later  made  to  isolate  the  strepto- 
coccus. In  general,  the  negative  character  of  the  findings  has 
no  significance.  There  is  one  particular  exception.  The  in- 
fluenza bacillus  has  been  kept  constantly  in  mind  and  our 
technique  has  been  suitable  to  grow  it.  We  have,  however, 
met  with  it  only  once  (Autopsy  1905,  82),  and  the  detail  of  that 
case  is  given. 

The  facts  which  concern  the  general  distribution  of  the  pneu- 
mococcus, the  constancy  with  which  it  occurs  in  the  pneumonic 
lung,  its  association  with  other  bacteria  in  the  pneumonic  lung, 
and  its  presence  in  the  lungs  of  persons  dying  of  other  causes 
than  acute  lobar  pneumonia  are  presented  in  the  following  table : 

(a)     Cases  in  which  the  pneumococcus  alone  was  isolated  from  the  lung : 

Acute  lobar  pneumonia 5  cases. 

Acute  pericarditis 2  cases. 

Acute  meningitis i  case. 

The  cases  of  pericarditis  and  meningitis  showed  areas  of  broncho-pneumonia. 


Charles  W.  Duval  and  Paul  A.  Lewis  75 

(6)     Cases  in  which  the  pneumococcus  was  isolated  together  with  the  strepto- 
coccus: 

Acute  lobar  pneumonia 22  cases. 

Wood-alcohol  poisoning i  case. 

Chronic  interstitial  nephritis i  case. 

The  cases  of  poisoning  and  of  nephritis  showed  no  lesion  of  the  lung. 

(c)  Cases  in  which  the  pneumococcus  was  isolated  together  with :   (i)  Strepto- 

coccus pyogenes  and  (2)   Pneumo-bacillus  of  Friedlander: 

Acute  lobar  pneumonia 4  cases. 

Cerebral  hemorrhage  with  broncho-pneumonia,    i  case. 

(d)  Cases  in  which  the  pneumococcus  was  isolated  together  with :   (i)  Strepto- 

coccus pyogenes,   (2)  Staphylococcus  aureus  and  albus,   (3)   Influenza 
bacillus,  (4)   Pseudo-diphtheria  bacillus: 

Acute  lobar  pneumonia i  case. 

(e)  Cases  in  which  the  pneumococcus  was  isolated  together  with :   ( i )  Strepto- 

coccus pyogenes,  (2)  Pneumo-bacillus  of  Friedlander,  (3)  Streptococcus 
mucosus : 

Acute  lobar  pneumonia i  case. 

After  the  work  was  well  under  way,  it  became  apparent  that 
the  pneumococcus  was  present  with  great  constancy  in  the 
pneumonic  lung,  and  that  it  often  occurred  in  the  healthy  as  well 
as  in  the  involved  portions  of  the  organ,  usually  in  association 
with  the  streptococcus.  It  was  realized  that  more  careful  study 
of  the  localization  of  pneumococci  and  streptococci  in  the  lungs 
of  pneumonia  cases  was  essential  to  a  consideration  of  the  rela- 
tionship of  these  micro-organisms  to  the  disease  and  to  one 
another.  To  answer  these  questions  the  examination  of  material 
from  the  lungs  of  patients  recently  dead  was  necessary.  De- 
cisive data  might  be  expected  from  cases  in  which  death  occurred 
in  an  early  stage  of  the  disease.  In  the  latter  part  of  the  winter 
we  have  had  two  cases  in  which  these  conditions  were  approxi- 
mated.    The  records  are  reported  in  detail. 

One  of  these  cases  (Autopsy  1905,  82)  was  unusual  in  that  B. 
influenzae  was  present  in  very  large  numbers.  From  its  peculiar 
interest  in  this  respect  the  clinical  history  is  reported. 

Case  I. — Acute  lobar  pneumonia.  Autopsy  1905,  47,  was  performed  nine 
hours  after  death.  There  was  late  red  and  early  gray  hepatization  of  the  right 
lower  lobe.  All  other  lobes  seemed  normal.  Twenty-six  plates  were  made  from 
the  upper,  middle,  and  lower  parts  of  each  lung;  i  %  glucose-agar  with  rabbit's 
blood  was  used  and  the  plates  were  seeded  on  the  surface.  Suspensions  of 
same  number  of  loops  of  material  from  each  portion  of  the  lung  were  employed. 


76 


Studies  on  the  Pneumococcus    ' 


All  the  plates  showed  some  colonies  of  Staphylococcus  aureus  and  all  showed 
the  pneumococcus  and  the  streptococcus  in  about  equal  numbers  as  estimated 
by  the  microscopic  examination.  Colonies  were  transplanted  and  cultiires 
identified  as  shown  in  the  accompanying  table. 


Lobe  of  Lung. 

Colonies. 

Pneumococcus. 

Streptococcus. 

Pneumonic  right  lower  lobe 

7 
3 
5 

2 

3 

4 
5 
3 
3 

2 

Healthy  right  upper  lobe 

"        right  middle  "    

"        left  lower        "    

"        left  upper        "    

Case  II. — E.  H.  B.,  aged  forty-five,  was  admitted  to  the  Boston  City  Hospital, 
May  3,  1905.  Clinical  diagnosis:  lobar  pneumonia.  On  admission  the  patient 
was  delirious.  A  member  of  his  family  stated  he  had  been  sick  for  one  week. 
There  was  considerable  prostration,  marked  cyanosis  and  dyspnoea.  He  was 
delirious  and  required  restraint.  The  eyes,  nose,  throat,  and  ears  were  negative. 
The  chest  was  symmetrical.  The  heart  sounds  were  indistinct  and  a  friction 
rub  was  heard  over  the  base  of  the  prscordium.  The  left  lung  revealed  dulness 
from  the  angle  of  scapula  to  the  base,  extending  into  the  axilla.  The  spleen  was 
not  felt.  The  abdomen  was  tympanic  but  not  tender.  The  temperature  on 
admission  was  104°,  the  pulse  135  and  respirations  40.  The  patient  remained 
deUrious,  requiring  restraint.  His  condition  became  progressively  worse  and 
death  ensued  on  the  second  day  after  admission. 

Autopsy  1905,  82,  was  performed  seven  hours  post-mortem.  Anatomical 
findings:  Acute  lobar  pneumonia;  acute  pleuritis;  acute  splenitis;  chronic 
pericarditis;  congestion  and  oedema  of  lungs.  Gross  examination  showed  a 
well-marked  consolidation  of  the  left  lower  lobe.  On  section  the  lower  two- 
thirds  of  the  pneumonic  lobe  were  found  in  the  stage  of  gray  hepatization. 
The  upper  third  was  moist,  granular,  and  dark  red  in  color.  The  pleura  was 
bathed  with  a  fibrino-purulent  exudate.  The  left  upper  lobe  showed  con- 
gestion and  oedema.  The  right  lung  was  negative.  Duplicate  smear-prepara- 
tions were  made  from  all  the  lobes.  One  of  each  was  stained  by  Gram's  method 
and  counter-stained  with  pyronin.  The  duplicates  were  stained  for  capsules. 
All  of  the  lobes  showed  encapsulated  diplococci.  The  smears  from  the  pneu- 
monic lobe  showed  comparatively  few  diplococci  contrasted  with  the  number 
from  the  normal  lobes.  In  all  the  smears  there  were  phagocytic  cells  which 
contained  from  two  to  sixteen  pair  of  Gram-positive  lanceolated  diplococci. 
Still  other  phagocytic  cells  contained  innumerable  Gram-negative  bacilli. 
Here  again  the  greater  numbers  were  observed  in  smears  made  from  the  un- 
involved  lobes. 


Charles  W.  Duval  and  Paul  A.  Lewis  77 

A  series  of  twenty-five  plates  was  seeded,  five  plates  of  uniform  seeding 
representing  each  lobe.  All  plates  developed  colonies  of  the  pneumococcus, 
Streptococcus  pyogenes,  B.  influenzse,  B.  pseudo-diphtheriee,  Staphylococcus 
albus  and  aureus.  The  number  of  colonies  of  pneumococcus  and  B.  influenzas 
was  greater  on  the  plates  representing  parts  of  the  lung  other  than  the  pneu- 
monic area.  The  colonies  of  B.  influenzae  ranged  from  one  to  three  hundred 
per  plate.  Though  all  the  plates  developed  colonies  of  Staphylococcus  aureus, 
no  one  plate  contained  more  than  fifty.  The  number  of  colonies  of  pseudo- 
diphtheria  bacillus  was  relatively  small.  Plates  were  also  made  from  the  heart's 
blood  (i  c.c.  of  blood  being  suspended  in  lo  c.c.  of  dilutant).  Here  again 
developed  pneumococcus,  streptococcus,  B.  influenzse,  B.  pseudo-diphtherise, 
and  staphylococcus.  A  few  plates  contained  so  many  colonies  of  B.  influenzae 
that  it  was  impossible  to  determine  their  number. 

From  the  study  of  these  two  cases  no  conclusions  are  war- 
ranted. 

II.  Surgical  Material. — The  results  of  the  examination  of 
surgical  material  can  be  summarized  briefly. 

Pneumococcus  present  in  pure  cultures : 

Alveolar  abscess i  case. 

Abscess  in  groin i  case. 

Abscess  in  chest i  case. 

Pelvic  abscess i  case. 

Septic  knee i  case. 

Septic  uterus 2  cases. 

Acute  mastoiditis 2  cases. 

Pneumococcus  and  streptococcus: 

Abscess  of  shoulder i  case. 

The  study  of  pleural  exudates  deserves  special  mention.  Twenty-five 
examinations  have  been  made. 

Pneumococcus  alone 9  cases. 

Pnetunococcus  and  streptococcus 6  cases. 

Streptococcus  alone 10  cases. 

It  is  noteworthy  that  in  the  early  examinations  it  was  con- 
sidered sufficient  to  identify  either  the  pneumococcus  or  the 
streptococcus,  and  no  consistent  effort  was  made  at  any  time  to 
grow  both  the  pneumococcus  and  the  streptococcus. 

III.  Cerebrospinal  Fluid. — From  the  cerebro-spinal  fluid 
taken  by  I1  unbar  puncture  from  two  cases,  the  pneumococcus  was 
isolated  in  pure  culture.  The  material  was  sent  from  outside  the 
hospital  and  no  subsequent  history  has  been  obtained. 

IV.  Sputum. — By  the  plate  method  the  pneumococcus  has 
been  isolated  from  the  sputum  of  several  cases  of  pneumonia. 


78  Studies  on  the  Pneumococcus 

Exarainations  of  sputa  from  cases  of  chronic  bronchitis  and 
suspected  tuberculosis  have  shown  the  presence  of  the  same 
organisms.  The  pneumococcus  was  isolated  eight  times  from 
tubes  inoculated  for  the  routine  examination  for  diphtheria  bacilli. 

Saliva  from  the  mouths  of  twenty-four  individuals  in  the 
laboratory  and  hospital  was  examined.  These  people  had  never 
had  pneumonia  and  were  in  perfect  general  health  at  the  time  of 
examination,  no  account  being  taken  of  the  slight  chronic  lesions 
of  the  pharynx  with  which  most  people  in  this  climate  are  af- 
fected in  the  winter  months.  Pneumococci  were  easily  isolated 
in  every  instance.  In  this  series  of  examinations  no  considera- 
tion was  given  to  cultures  which  did  not  ferment  inulin. 

Saliva  was  suspended  in  bouillon  and  plated  as  above  de- 
scribed. Colonies  were  transferred  to  blood-agar  tubes.  Stains 
were  made  from  the  growth  twenty-four  hours  old  by  Gram's 
method  and  usually  by  methods  for  identification  of  capsules. 
If  the  morphology  and  growth  were  characteristic,  the  cultures 
were  at  once  tested  in  inulin  bouillon  and,  as  before  stated,  only 
those  producing  a  marked  acidity,  shown  by  titration,  have 
been  regarded  as  pneumococci.  As  there  is  no  evidence  that 
the  streptococcus  ever  ferments  inulin,  these  points  seem  suf- 
ficiently conclusive  for  the  present  purpose.  Inulin-splitting 
pnetmiococci  we  conclude  are  present  during  the  winter  season 
in  the  saliva  of  practically  every  person  living  under  ordinary 
city  conditions. 

Our  studies  on  the  distribution  of  the  pneumococcus  have 
shown  that  it  is  to  be  found  practically  always  at  autopsy  in  the 
lungs  of  people  dying  with  lobar  pneumonia,  and  that  it  is  found 
in  the  healthy  as  well  as  in  the  diseased  portions  of  the  lung. 
At  times  this  micro-organism  is  found  in  the  non-pneiunonic 
lungs  of  persons  dying  from  causes  other  than  pneumonia. 
Very  frequently  the  pneumococcus  is  found  in  purulent  affec- 
tions of  the  serous  cavities  and  in  acute  abscesses  of  other  parts. 
In  the  mouth  and  throat  of  persons  in  good  general  health  it 
is  found  so  often  that  its  presence  can  have  no  pathological 
significance. 

It  is  further  evident  that  Streptococcus  pyogenes  and  the 


Charles  W.  Duval  and  Paul  A.  Lewis  79 

pneumococcus  are  found  in  much  the  same  places  and  con- 
ditions and  very  often  occur  together.  The  facts  of  general 
distribution  hardly  warrant  the  supposition  that  the  one  bac- 
terium has  a  closer  etiological  relation  to  acute  lobar  pneumonia 
than  the  other.  A  more  extended  and  careful  study  of  the 
local  distribution  of  these  two  organisms  in  pneumonic  lungs 
might,  however,  lead  to  valid  negative  or  positive  conclusions 
on  this  most  important  point. 

CHARACTERISTICS    OF    THE    PNEUMOCOCCUS    IN    RELATION    TO    ITS 

SOURCE    OF    ORIGIN. 

In  attempting  to  draw  distinctions  between  pneumococci  of 
various  sources  we  have  met  with  no  success.  We  have  paid 
most  attention  to  cultural  features.  The  fermentation  of  inulin 
characterizes  a  definite  group  of  pneimiococci.  Inulin-splitting 
pneumococci  are  found  with  frequency  among  those  cultivated 
froin  all  sources.  We  have  found  no  other  cultural  feature  which 
is  more  distinctive. 

We  have  also  considered  the  virulence  of  cultures  to  some  ex- 
tent. We  have  tested  the  virulence  of  thirty-five  recently 
isolated  cultures,  in  large  part  from  the  pneumonic  lung.  We 
have  used  young  rabbits.  The  dosage  has  been  either  lo  c.c. 
of  a  twenty-four-hour  glucose-bouillon  culture  showing  heavy 
growth  or  the  whole  of  a  twenty-four-hour  culture  on  glucose 
agar  plus  rabbit's  blood.  In  some  instances  two  or  three  such 
agar  cultures  have  been  given.  The  injection  has  usually  been 
made  into  the  peritonetim.  For  control,  animals  have  been 
injected  subcutaneously  and  intravenously.  In  only  one  in- 
stance has  a  fatal  result  been  attributable  directly  to  the  culture ; 
the  organism  was  isolated  from  the  saliva  of  a  healthy  person. 
We  do  not  consider  our  work  on  these  points  sufficiently  ex- 
tensive to  be  entirely  conclusive;  so  far  as  we  have  gone,  our 
cultures  from  all  sources  have  been  identical  in  possessing  a  low 
grade  of  virulence  towards  laboratory  animals  and  in  showing 
no  cultural  variation  coincident  with  their  source. 

As  bearing  on  the  results  of  previous  workers  who  have  written 


80  Studies  on  the  Pneumococcus 

on  the  virulence  of  the  pneumococcus,  we  offer  the  following  ob- 
servations. Depending  on  the  plate  method  for  isolation,  we  have 
frequently  inoculated  into  animals  the  original  material,  namely, 
sputum,  pus,  or  suspensions  from  the  lung.  The  results  of  these 
inoculations  fall  into  two  distinct  classes.  In  several  instances 
the  pneumococcus  was  present  in  pure  culture  as  estimated  by 
smear  preparations  and  plate  culture.  In  each  of  these  cases 
the  animals  survived  the  injection  of  large  quantities  of  material. 
One  young  rabbit  received  5  c.c.  of  pus  from  the  base  of  the 
brain  in  a  case  of  pneumococcus  meningitis.  The  examination 
of  smear  preparations  showed  large  numbers  of  encapsulated 
pneumococci  in  a  good  state  of  preservation.  Two  of  these 
animals  still  live.  Two  were  killed  on  the  fifth  day  and  the 
pneumococcus  was  recovered  from  their  blood.^ 

In  the  other  class  of  cases  the  material  examined  contained 
a  mixture  of  bacteria.  The  injection  of  small  or  moderate 
doses  gave  in  most  instances  a  fatal  result  in  from  one  to  four 
days.  The  pneumococcus  was  always  found  in  the  blood,  gener- 
ally in  pure  culture.  If  bacilli  belonging  to  the  Friedlander 
group  were  present,  they  were  frequently  found  in  the  blood  of 
the  inoculated  animal,  especially  when  the  guinea-pig  was  used. 
In  view  of  the  series  of  experiments  with  material  containing  the 
pneumococcus  only,  and  the  consistently  negative  results  with 
pure  cultures,  we  doubt  whether  the  presence  of  the  pneumo- 
coccus in  the  blood  of  these  animals  indicates  that  it  alone  is 
responsible  for  the  death  of  the  animal. 

It  seems  probable  that  when  the  blood  of  the  animal  first 
inoculated  is  transferred  directly  to  another  animal,  a  false  im- 
pression of  the  virulence  of  the  culture  finally  recovered  may  be 
given,  because  of  the  exalted  virulence  attained  by  the  direct 
passage  from  animal  to  animal. 

THE    ACTION    OF    PNEUMOCOCCI    ON    INULIN. 

Early  in  the  year  we  placed  great  reliance  on  the  inulin  test 
as  an  absolute  means  of  differentiating  the  pneumococcus  from 

3  The  culture  isolated  from  the  rabbit  inoculated  with  pus  frora  this  case 
of  meningitis  is  described  in  more  detail  in  our  discussion  of  the  non-inulin- 
fermenting  pneumococci. 


Charles  W.  Duval  and  Paul  A.  Lewis  81 

Streptococcus  pyogenes.  We  used  then  the  senim-water-inulin 
medium  proposed  by  Hiss.  Several  hundred  tubes  of  the  serum- 
water  medium  made  with  inulin  ("Merck")  and  beef -serum 
gave  consistent  results  with  ten  pneumococcus  cultures  and 
several  cultures  of  Streptococcus  pyogenes. 

These  cultures  have  been  retained  and  have  served  as  types 
with  which  our  organisms  isolated  later  have  been  compared. 
Except  for  the  one  lot  of  inulin-serum-water  medium  mentioned, 
which  fortunately  was  large,  we  have  had  nothing  but  failure  in 
its  use.  This  failure  we  have  been  unable  to  explain  satis- 
factorily. We  do  not  feel  convinced  that  the  medium  is  essen- 
tially faulty,  but  there  has  been  perhaps  some  disturbing  factor 
in  our  material  or  method  of  preparation  which  we  have  not 
been  able  to  discover  or  control.  We  have,  however,  had  no 
trouble  in  obtaining  constant  results  with  the  pneumococcus  in 
serum-water  media  containing  other  carbohydrates. 

In  trying  to  explain  the  inconstancy  of  our  results  we  have 
proceeded  as  follows:  The  reaction  of  the  medium  has  been 
carefully  controlled  and  varied  from  neutral  to  i  %  acid  to 
phenolphthalein  with  differences  of  .i  %  of  acid.  These  differ- 
ences bring  about  distinct  variations  in  the  opacity  of  the  prep- 
aration. But  when  inulin  is  added  no  one  degree  of  acidity  is 
found  to  give  more  consistent  results  than  another.  Inulin  has 
been  sterilized  separately  and  added  to  the  tubes  with  a  pipette, 
but  the  results  have  been  no  better.  We  have  worked  with  the 
following  preparations  of  inulin : 

Merck's  inulin,  white. 

"        alant  starch,  Lot  I. 

"  "  "       white,  Lot  II. 

"        inulin  ("highest  purity"). 
Bausch  and  Lomb's  inulin,  white. 

The  preparation  which  gave  the  one  consistent  result  was 
Merck's,  Lot  II.  Repeated  trials  with  the  same  lot  have  failed. 
The  two  lots  of  Merck's  white  inulin  and  Bausch  and  Lomb's 
white  inulin  when  tested  in  lo  %  solution  in  distilled  water  with 
Fehling's  solution  have  shown  reducing  substances  present  in 
quantity  equivalent  to  from  2  to  3  %  of  dextrose  in  the  dry 


82  Studies  on  the  Pneumococcus 

substance.  If  a  lo  %  solution  in  distilled  water  is  subjected  to 
sterilization  in  the  autoclave  (at  lo  lbs.  pressure  for  ten  minutes) , 
the  percentage  of  reducing  substances  may  be  increased  to  five. 
These  reducing  substances  may  not  be  dextrose  and  they  may 
indicate  the  presence  of  other  impurities  which  do  not  reduce 
the  copper.  One  small  quantity  of  inulin  (Kiliani)  obtained 
from  Bausch  and  Lomb  was  subjected  to  the  autoclave  before 
the  possibility  of  injury  had  suggested  itself.  Subsequently  it 
was  found  to  contain  about  the  same  percentage  of  the  reducing 
substances  as  that  shown  by  other  preparations.  This  prepara- 
tion we  have  not  been  able  to  buy  again. 

INULIN    BOUILLON. 

In  the  hope  of  avoiding  the  difficulties  encountered  in  the  use 
of  the  inulin-serum-water  medium,  at  the  suggestion  of  Dr. 
Theobald  Smith  we  undertook  a  series  of  experiments  with 
inulin  in  bouillon.  The  outcome  exceeded  our  expectations. 
The  results  of  successive  titrations  remained  uniform,  and  we 
can  recommend  this  modification  of  the  inulin  test  for  routine 
work.  Using  these  results  as  a  basis,  pneumococci  can  be 
classified  as  follows : 

r  I.  High  acid  producers. 

A.  Inulin  fermenters    <  II.  Medium  acid  producers. 

(  III.  Low  acid  producers. 

B.  Inulin  non-fermenters. 

Bouillon  is  made  according  to  standard  methods  from  beef.  It 
is  reduced  in  acidity  to  from  .2  to  .4  %  normal  to  phenolphtha- 
lein.  Subsequent  sterilization  restores  the  acidity  to  from  .5  to 
.8  %.  Carefully  cleaned  tubes  containing  9  c.c.  of  bouillon  are 
sterilized.  A  10  %  solution  of  inulin  in  distilled  water  is  ster- 
ilized in  the  autoclave  at  15  lbs.  pressure  for  fifteen  minutes, 
and  with  a  sterile  pipette  i  c.c.  of  the  inulin  solution  is  added  to 
each  tube  of  bouillon. 

The  tubes  are  simultaneously  inoculated  with  the  cultiires  to 
be  tested,  incubated  for  the  period  of  the  experiment,  and  then 
steamed  for  a  short  time  in  an  Arnold  sterilizer.  They  are  then 
titrated  for  percentage  acidity,  using  the  same  solution  and  in- 


Charles  W.  Duval  and  Paul  A.  Lewis  83 

dicator.  Theoretically  it  might  be  better  to  prepare  as  a  culture 
base  sugar-free  bouillon.  It  has  proved  satisfactory,  however, 
to  use  as  control  cultures  in  bouillon  containing  no  inulin. 

The  table  on  page  486  shows  the  result  of  titrations  in  two 
lots  of  inulin-bouillon  made  from  different  material  and  different 
brands  of  inulin.  The  period  of  growth  in  this  experiment  is  one 
week.  With  a  little  experience  the  high  and  medium  acid-pro- 
ducing organisms  can  be  distinguished  in  twenty -four  hours,  and 
the  low  acid-producing  organisms  in  forty-eight  hours,  although 
the  numerical  differences  at  these  periods  are  small  and  incon- 
stant. It  is  probable  that  if  the  number  of  cultures  worked  with 
were  doubled  or  tripled,  organisms  intermediate  between  these 
groups  and  types  might  be  found,  but  the  types  are  sufficiently 
marked  to  illustrate  the  efficiency  of  the  method  employed. 
With  two  or  three  exceptions,  all  of  our  inulin-fermenting  or- 
ganisms that  have  survived  prolonged  cultivation  appear  in  the 
table.  The  non-inulin-fermenting  group  might  be  greatty  en- 
larged by  the  addition  of  about  thirty  cultures  which  we  still 
retain.  As  the  differentiation  of  many  of  these  ctdtures  from 
Streptococcus  pyogenes  is  difficult  and  uncertain,  we  prefer  to 
test  only  those  whose  identity  rests  on  secure  ground. 

The  classification  is  based  on  the  reactions  of  cultures  which 
have  been  several  weeks  under  cultivation.  We  have  recently 
made  a  number  of  titrations  with  freshly  isolated  cultures. 
Many  of  them  have  given  reactions  of  from  5.8  to  6.1  %  of  a 
normal  acid  solution  at  the  end  of  a  week.  We  should  hesitate, 
therefore,  to  separate  the  two  cultures  of  Type  I  from  those  of 
Type  II  on  this  basis  alone.  As  they  are  the  only  cultures 
among  our  stock  which  ferment  mannit,  and  as  their  titration  is 
always  somewhat  higher  than  that  of  any  other  culture,  we  feel 
that  the  distinction  is  justified.  The  cultures  of  Group  A  we 
think  are  all  pneumococci. 

Group  B  requires  explanation.  By  comparison  of  the  figures 
given  for  the  plain  bouillon  controls  with  those  given  for  the 
members  of  Type  IV,  it  is  seen  that  the  average  of  the  latter  is 
somewhat  higher.  This  is  probably  due  to  the  presence  of  small 
quantities  of  fermentable  impurities  in  the  inulin.     Inspection 


84  Studies  on  the  Pneumococcus 

GROUP  A.=  Inulin  Fermented. 


i-i 

o 
.a 

5 

2 
o 

Total  Acidity 

> 

<; 

Remarks. 

Bouillon. 

Type. 

Plain. 

Inulin 
Lot  I. 

Inulin 
Lot  II. 

(Uninoculated  Controls  Titrate 
at  0.6  to  0.8  %.) 

I 

CD. 

i6 

1.1% 

6.5% 

6.8% 

These     cultures     ferment 

High    Acid 

CD. 

12 

1.0% 

6.6% 

6.4% 

6.S7  % 

mannit. 

Producers 

II 

CD. 

9 

.9% 

4.3  % 

4.3% 

The  culttires  of  this  type 

CD. 

19 

i.o% 

4 

3% 

4.2% 

and  also  those  of  Type  I 

Medium 

CD. 

20 

1.1% 

4 

2% 

4.6% 

are    distinguished    from 

Acid 

CD. 

21 

1.0% 

4 

9% 

5.0% 

4.53  % 

Type  III  and  the  non-fer- 

Producers 

CD. 

22 

.9% 

4 

5% 

4.8% 

menters  by  the  fact  that 

CD. 

23 

.9% 

4 

5% 

4.6% 

they   produce    a   diffuse 

CD. 

24 

1.1% 

4 

8% 

4.5  % 

cloud    and    heavy    sedi- 

ment   in    the    bouillon, 

recognizable  in  from  16 

to  24  hours. 

CD. 

I 

1.0% 

2.6  % 

2.6% 

These  cultures  are  also  to 

III 

CD. 

2 

1.1% 

2.3% 

2.7% 

be     distinguished     from 

CD. 

4 

1.1% 

2.8% 

2.3  % 

the  non-fermenters  by  a 

CD. 

6 

1-2% 

2.6% 

2.4% 

slight  diffuse  clouding  of 

Low  Acid 

CD. 

8 

.9% 

3.3  % 

2.2% 

2.6% 

the    bouillon    which    is 

Producers 

CD. 

10 

1.0% 

2.6% 

2.7% 

marked    on   the    second 

CD. 

18 

1.1% 

Z-Z  % 

2.6% 

day  of  cultivation. 

CD. 

25 

.9% 

2.0% 

1.9% 

CD. 

26 

1.1% 

3 

0% 

3.1% 

GROUP  B.  =  Inulin  not  Fermented. 


IV 


CD.     7 

.9% 

1.4% 

1-5% 

CD.  27 

.8% 

1.6% 

1.4% 

CD.  14 

1.0% 

1.1% 

1.5% 

CD.  28 

1.1% 

1.6% 

1.7% 

1.65  % 

CD.  29 

.9% 

1.7% 

1.2% 

CD.  13 

1.0% 

1.4% 

1-3% 

These  cultiures  have  been 
fully  identified.  The 
complete  description  of 
CD.  7  appears  in  the 
text,  page  487. 

This  group  could  be 
greatly  enlarged,  but  cul- 
ttires might  be  intro- 
duced that  could  not  be 
certainly  differentiated 
from  Streptococcus  py- 
ogenes.    See  page  485. 


Note:    Streptococcus  pyogenes  cultures  titrate  up  to  1.6  %  acid. 


Charles  W.  Duval  and  Paul  A.  Lewis  85 

of  the  titrations  with  this  group  shows  that  the  method  gives  no 
aid  in  the  identification  of  these  ctdtures.  The  cultures  included 
in  the  table,  judged  by  all  of  their  characters,  are  without 
doubt  pneumococci.  We  believe,  however,  that  some  cultures 
which  according  to  the  titration  with  inulin  bouillon  belong  in 
this  group  are  typical  streptococci.  It  is  not  our  purpose  at 
present  to  classify  the  cultures  of  this  group.  In  order  to  make 
clear  our  point,  that  there  are  pneumococci  which  do  not  ferment 
inulin,  we  give  the  following  description  and  history  of  such  a 
culture. 

Culture  C.  D.  j. — Isolated  by  plate  method  in  December,  1904,  from  the  Iting 
of  a  case  of  acute  lobar  pneumonia.  A  large  number  of  colonies  were  examined, 
but  no  inulin  fermenter  was  found.  C.  D.  7  was  at  that  time  a  Gram-positive 
diplococcus.  The  lanceolate  pairs  were  surrounded  by  a  large  capsule  which 
could  be  easily  stained  by  either  of  the  Hiss  methods.  The  capsule  persisted 
for  some  weeks  in  culture,  but  was  finally  lost.  The  colonies  on  Loeflfler's 
blood-serum  and  rabbit's  blood-agar  were  large  and  moist.  Growth  in  glucose- 
bomllon  was  profuse,  the  medium  being  diffusely  clouded.  Litmus  milk  was 
coagulated  in  twenty-four  hours,  and  in  forty-eight  hours  the  litmus  in  the 
lower  half  of  the  tube  was  decolorized.  Lactose-serum  water  was  coagulated 
in  twenty-four  hours. 

At  the  present  time  (June  20,  1905)  these  characters,  excepting  the  capsule, 
are  well  preserved.  Glucose-bouillon  is  still  diffusely  clouded,  and  a  Gram 
stain  made  from  a  bouillon  tube  shows  a  predominant  number  of  rather  asym- 
metrical pairs  of  large  lanceolate  cocci.  There  are  many  chains,  but  they  are 
all  short,  containing  from  four  to  ten  cocci.  The  morphological  characteristics 
are  intact,  and  as  well  marked  as  at  the  time  of  isolation.  This  culture  has 
never  coagulated  the  serum-water  medium,  and  in  oiir  titrations  with  iniilin- 
bouillon  its  total  acidity  has  never  gone  above  1.5  %  acid. 

Although  we  have  given  to  few  cultures  among  the  non-fer- 
menting group  so  close  attention  as  we  have  to  C.  D.  7,  we  are 
certain  that  others  of  these  cultures  are  pneumococci  and  that 
in  routine  examinations  the  non-inulin-fermenting  group  will 
be  frequently  encountered.  Since  the  introduction  of  inulin 
has  made  the  identification  of  some  groups  of  pneimiococci 
relatively  easy,  the  difficulties  previously  encountered  in  dis- 
tinguishing this  organism  from  Streptococcus  pyogenes  are 
significant  only  in  the  non-inulin-fermenting  group.  The  cul- 
tures which  ferment  inulin,  and  a  few  of  those  which  do  not, 
have  the  pneumococcus  characters  so  well  developed  and  preserve 


86  Studies  on  the  Pneumococcus 

them  so  constantly  in  culture  that  their  identification  by  means 
formerly  available  is  not  very  difficult.  On  the  other  hand, 
it  is  probable  that  carefiil  study  of  the  non-fermenting  group 
will  establish  intermediary  varieties  and  may  render  unavoid- 
able the  conclusion  that  the  pneumococcus  and  the  strepto- 
coccus are  races  of  a  single  species  of  bacterium.  That  the 
pneumococcus  and  streptococcus  are  not  the  extreme  types  of 
such  a  species  seems  not  improbable  if  one  takes  into  considera- 
tion the  saprophytic  streptococcus  on  the  one  hand  and  such 
cultures  as  the  encapsulated  diplococcus  of  which  we  will  give 
an  account  on  the  other.  There  may  also  be  varieties  of  the 
species  which  have  been  separated  even  farther  from  the  usual 
types.  For  example,  we  will  give  in  detail  the  record  of  a  cul- 
ture previously  mentioned : 

Culture  from  case  of  cerebrospinal  meningitis  (Autopsy  1905,  t,^)- — Stained 
preparations  from  the  pus  at  the  base  of  brain  showed  large  numbers  of  en- 
capsulated diplococci  staining  by  Gram's  method.  Cultiires  on  LoefHer's 
blood-serum  and  blood-agar  were  negative  so  far  as  micro-organisms  other  than 
pneumococci  were  concerned.  There  was  no  growth  on  the  surface,  but  careful 
examination  of  the  condensation  water  showed  a  few  encapsulated  Gram- 
staining  diplococci.  Subcultures  were  negative.  Five  cubic  centimeters  of 
the  pus  at  the  base  of  the  brain  were  inoculated  into  the  peritoneal  cavity  and 
subcutaneous  tissues  of  a  rabbit  weighing  750  grams.  After  twenty-four  hours, 
the  animal  ate  well  and  had  lost  no  weight.  On  the  fifth  day  it  was  killed.  Ex- 
amination of  the  heart's  blood  showed  a  few  lanceolate  diplococci  retaining 
Gram's  stain  and  possessing  a  distinct  capsule.  Cultures  on  LoefHer's  blood- 
serum  were  negative.  In  the  condensation  water  of  blood-agar  tubes,  a  few 
diplococci  agreeing  in  negative  and  positive  character  with  those  from  the 
original  material  were  found.  After  repeated  and  frequent  subcultvires  cover- 
ing a  period  of  six  weeks,  during  which  time  proliferation  was  very  slight  and 
confined  to  the  condensation  water,  the  diplococcus  rather  suddenly  increased 
its  activity  of  growth.  It  grew  fairly  well  in  fine  colonies  on  the  surface  of 
blood-agar,  but  did  not  grow  on  plain  agar  or  on  Loeffier's  blood-serum.  In 
milk  it  produced  a  slight  acidity  after  several  days.  No  fermentation  of  sugars 
could  be  demonstrated  in  the  serum-water-media.  After  two  or  three  genera- 
tions of  this  relatively  abundant  growth  its  activity  again  declined,  and  in  spite 
of  repeated  efforts  the  culture  died  out.  Cultivation  failed  though  the  reaction 
and  sugar  content  of  media  were  varied  and  blood  and  serum  were  added. 

The  organism  just  described  was  evidently  a  pneumococcus 
which  differed  very  widely  from  the  members  of  this  group 
usually  encountered.    It  had  morphological  characteristics  which, 


Charles  W.  Duval  and  Paul  A.  Lewis  87 

as  shown  in  Fig.  5,  were  sufficiently  definite  to  classify  it  as 
pneumococcus.  Perhaps  one  could  conclude  from  the  experi- 
ence with  this  culture  that  there  are  parasitic  pneumococci  so 
delicate  as  to  be  unrecognized  by  our  present  methods  of  cul- 
tivation and  at  the  same  time  possessing  possibly  pathological 
significance. 

It  might  be  said  that  the  method  which  we  propose  for  using 
inulin  is  too  tedious  for  routine  work.  To  obviate  this  difficulty, 
we  have  made  control  observations  with  inulin-bouillon  made 
in  bulk  and  sterilized  after  tubing.  The  quantity  in  each  tube 
need  not  be  the  same,  and  the  medium  is  as  efficient  as  that  more 
carefully  made.  The  inulin  when  sterilized  in  the  bouillon  does 
not  undergo  sufficient  reduction  to  injure  the  efficiency  of  the 
test.  The  time  we  have  used  for  the  reported  tests  (one  week) 
is  necessary  to  bring  out  the  details  of  grouping  which  we  have 
described,  but,  as  has  already  been  mentioned,  organisms  be- 
longing to  different  groups  can  be  roughly  separated  in  a  shorter 
time.  Neither  is  the  titration  a  necessity;  the  medium  and 
high  acid-producing  subdivisions  cause  in  the  inulin-bouillon  a 
diffuse  cloud  and  heavy  sediment  which  are  apparent  in  from 
sixteen  to  twenty-four  hours.  The  low  acid-producers  show  a 
well-marked  diffuse  cloud  in  the  medium  on  the  second  day. 
The  non-fermenters  never  cloud  the  bouillon  and  the  sediment 
of  bacterial  growth  remains  slight.  Though  titration  is  not 
necessary  for  the  recognition  of  the  fermenting  pneumococci,  it 
has  served  to  distingtdsh  finer  differences  between  groups,  and 
as  a  routine  optical  method  the  bouillon  has  all  the  advantages 
of  the  serum- water  medium.  We  think  that  the  group  of  low 
acid-producers  cause  coagulation  irregularly,  because  the  total 
acidity  which  they  produce  under  unfavorable  conditions  is 
often  just  sufficient  to  change  litmus,  and  this  degree  of  acidity 
seems  to  approximate  the  coagulation  point  of  the  serum- water. 
The  inulin-bouillon  has  the  added  advantage  that  its  constituents 
are  easily  procured  and  combined,  its  constitution  is  more  uni- 
form, and  the  reaction  produced  is  more  delicate  when  estimated 
by  titration  for  percentage  of  acidity  than  is  the  case  with  the 
serum-water  medium. 


88  Studies  on  the  Pneumococcus 

We  have  retested  the  samples  of  inulin  before  used,  and  find 
that  when  used  in  the  bouillon  the  brands  labeled  "Highest 
purity,"  which  are  hard  to  secure,  are  not  essential.  All  the 
samples  of  white  inulin  mentioned  before  and  one  lot  from 
another  supply  house  act  equally  well. 

The  irregularity  of  the  results  obtained  by  us  with  inulin- 
serum-water  medium  has  not  been  satisfactorily  explained. 
Since  the  difference  in  favor  of  the  bouillon  might  be  referred  to 
a  greater  quantitative  development  of  bacteria  in  this  medium, 
we  have  tested  media  with  three  actively-growing  type  cultures 
of  the  pneumococcus,  varying  in  their  fermentative  activity, 
and  with  one  culture  of  Streptococcus  pyogenes.  We  have 
estimated  by  plating  after  twenty-four  hours  of  growth  the 
ntimber  of  bacteria  in  plain  bouillon,  plain  serum- water,  dex- 
trose-bouillon, dextrose-serum- water,  inulin-bouillon,  and  inuHn- 
sertim- water.  There  was  no  difference  in  the  number  of  colonies 
developing  on  the  plates  from  the  bouillon  media  and  those  from 
the  serum- water  media. 

Our  conclusions  with  regard  to  the  inulin  test  are  as  follows : 

I.  The  routine  isolation  of  the  pneimiococcus  is  much  facili- 
tated by  this  test.  Until  the  number  and  pathological  signifi- 
cance of  the  non-inuHn-fermenting  group  of  pneumococci  is 
established,  the  test  must  be  carefully  controlled  by  the  older 
methods  of  identification. 

II.  A  final  opinion  as  to  the  best  method  of  using  the  inulin 
cannot  be  offered.  We  believe,  however,  that  the  inulin-bouillon 
method  by  mere  inspection  is  as  easy  to  carry  out  and  as  accurate 
as  the  serum-water  method,  and  that  with  careful  titrations  it  is 
more  delicate  as  it  offers  a  means  of  subdividing  the  inulin-fer- 
menting  pneumococci  into  definite  groups — a  differentiation 
impossible  by  the  latter. 

STREPTOCOCCUS    MUCOSUS. 

In  the  course  of  the  investigation  we  have  twice  encountered 
a  Gram-staining,  encapsulated  diplococcus  with  characteristics 
which  make  it  peculiarly  interesting.  We  have  identified  it 
with  the  diplococcus  described  by  Richardson,  and  by  comparison 


Charles  W.  Duval  and  Paul  A.  Lewis  89 

of  cultures  with  the  coccus  described  by  Howard  and  Perkins. 
As  we  have  noted  some  points  in  its  morphology,  cultural  features, 
and  pathogenicity  which  do  not  appear  in  their  descriptions,  we 
will  describe  the  organism  in  some  detail. 

Source. — The  first  of  our  cultures  of  this  micrococcus  was 
isolated  from  the  lung  of  a  case  of  acute  lobar  pneumonia.  The 
lung  showed  gray  hepatization.  Our  description  is  based  on 
the  study  of  this  culture.  Recently  a  second  organism  which 
has  the  same  cultural  features  has  been  obtained  from  the  pus 
from  a  mastoid  abscess. 

Morphology. — Morphologically  this  micro-organism  is  an  en- 
capsulated, lanceolate  diplococcus.  It  retains  Gram's  stain. 
Its  capsule  persists  in  cultures  and  is  readily  stained  by  any 
capsule  method ;  it  is  frequently  stained  by  the  ordinary  staining 
methods.  Fig.  i,  for  example,  shows  a  stained  preparation  made 
according  to  Gram  from  a  culture  on  blood-agar.  In  the  prepara- 
tion, capsules  were  not  noticed,  but  the  photograph  shows  them 
faintly  stained.  Figs.  2 , 3,  and  4  are  from  preparations  also  stained 
by  the  m^ethod  of  Gram  slightly  modified.  Smear  preparations  on 
clean  slides  are  made  from  the  exudate  of  an  animal  recently  dead 
after  inoculation  with  the  culture.  After  drying  in  the  air  they  are 
thoroughly  fixed  in  the  flame.  While  the  slide  is  still  hot  enough 
to  cause  the  stain  to  steam,  the  preparation  is  covered  with  the 
solution  of  gentian- violet,  which  is  left  on  the  slide  for  one  or 
two  minutes.  lodin  is  applied  in  the  usual  way,  but  decoloriza- 
tion  with  alcohol  is  not  completed.  The  thin  parts  of  the  smear 
are  completely  washed  out,  the  thicker  parts  are  left  stained. 
Counterstaining  is  omitted.  Some  fields  will  now  be  found  in 
which  there  is  a  sharp  differentiation  of  the  cocci  and  in  which 
the  capsule  as  well  has  retained  the  blue.  The  results  are  in- 
constant and  it  may  be  necessary  to  make  many  preparations 
before  a  good  one  is  obtained.  The  result  when  successful  is 
a  sharp  picture  in  which  the  details  of  the  capsule  and  its  relation 
to  the  coccus  are  better  shown  than  by  other  methods.  By  the 
study  of  these  preparations,  appearances  which  we  have  en- 
deavored to  show  by  photograph  can  be  made  out.  Fig.  2  (a) 
shows  a  single  coccus  surrounded  by  a  complete  circular  capsule. 


90  Studies  on  the  Pneuraococcus 

Fig.  2  (6)  shoAvs  a  pair  of  small  cocci  with  a  poorly  defined  line  of 
demarcation  between  them.  On  opposite  sides  of  the  capsule 
and  in  the  line  of  the  transverse  division  between  the  individuals 
of  the  pair  are  a  sHght  thickening  of  the  capsule  and  a  thin 
Hnear  projection  toward  the  center  of  the  pair.  At  d  in 
Fig.  2  and  at  c  in  Fig.  3  these  lateral  thickenings  have 
increased  in  breadth  and  inward  extension.  The  dividing 
line  between  the  cocci  is  better  marked.  At  e,  Fig.  4,  the 
division  between  cocci  is  complete  and  the  capsule  surrotinds 
both  elements. 

The  capsules  can  also  be  stained  by  other  methods.  If 
Wright's  modification  of  the  Romano wsky  stain  ibe  used,  the 
cocci  take  a  blue  stain  and  the  capsular  portion  takes  the  pink 
color.  The  outline  of  the  capsule  is  distinct  and  the  points  that 
we  have  endeavored  to  illustrate  are  brought  out  with  greater 
deHcacy. 

Thionin  blue  in  saturated  aqueous  solution  stains  the  cocci 
and  capsule  from  an  exudate  rather  lightly.  The  stain  must 
be  steamed  for  a  few  moments  and  is  then  washed  off  with 
water.  The  capsule  is  delicately  outlined  in  purple  and  the  de- 
tails before  described  are  seen.  The  preparations  made  by  these 
later  methods  are  less  stdtable  for  illustration  than  those  by 
Gram's  method. 

The  capsules  stain  by  either  of  the  methods  of  Hiss,  but  the 
details  here  described  are  rarely  brought  out  by  them,  the 
capsule  appearing  filled  with  the  stain  rather  than  outlined 
by  it. 

The  capsule  of  this  micrococcus  is  always  present  when  the* 
organism  is  growing  and  moist.  It  can  be  readily  demonstrated 
by  any  of  the  special  methods  of  capsule  staining  and  with  almost 
equal  readiness  by  methods  which  do  not  ordinarily  demonstrate 
capsules  on  the  pneumococcus  and  streptococcus.  In  its  de- 
velopment and  division  the  capsule  follows  closely  the  division 
and  separation  of  the  coccus.  By  the  modified  Romanowsky 
stain  the  basophilic  coccus  can  be  well  differentiated  from  the 
oxyphilic  capsule.  The  capsule  of  this  diplococcus  is  evidently 
more  highly  developed  and  difterentiated  than  that  of  the  pneu- 


Charles  W.  Daval  and  Paul  A.  Lewis  91 

mococcus.  In  these  facts  there  is  some  evidence  that  the  capsule 
of  this  micrococcus  may  be  a  part  of  the  bacterial  cell.  Possibly 
it  is  the  precursor  of  the  cytoplasm  of  the  more  highly  developed 
unicelltilar  plants. 

Cultural  Features. — The  micrococcus  grows  well  with  a  profuse, 
very  watery  growth  on  the  surface  of  i  %  glucose-agar,  .5  % 
acid  to  phenolphthalein,  if  about  i  %  of  "fresh"  defibrinated 
blood  be  added.  The  individual  colonies  are  perfectly  trans- 
parent and  attain  in  forty-eight  hours  a  size  6  mm.  in  diameter. 
It  also  grows  in  a  glucose-agar  stab  culture  without  blood  if  the 
medium  is  made  from  beef  and  is  suitable  in  reaction.  In  this 
way  there  can  be  developed  peculiar  and,  so  far  as  our  experience 
goes,  characteristic  wing-like  lateral  out-growths.  This  feature 
was  noted  by  Richardson,  and  together  with  the  persistence  of  the 
capsules  in  culture  serves  to  identify  his  culture  with  those  that 
we  have  isolated.  In  gelatin-agar  mixtures  of  proper  reaction 
and  sugar  content  growth  is  good,  but  lateral  outgrowths  in  stab 
cultures  do  not  appear.  Abundant  growth  can  be  obtained  on 
any  of  the  usual  solid  or  fluid  media  if  .5  c.c.  of  fresh  blood  be 
added  to  each  tube.  The  published  data  on  the  fermentation 
reactions  are  scanty.  Hiss  states  that  the  organism  of 
Howard  and  Perkins  ferments  inulin.  We  find  that  if  fresh  de- 
fibrinated blood  be  added  to  tubes  of  serum-water  medium  con- 
taining dextrose,  lactose,  maltose,  galactose,  mannit,  dextrin,  or 
inulin,  these  organisms  coagulate  the  medium  in  from  twenty- 
four  to  forty-eight  hours.  Controls  of  plain  serum- water  plus 
blood  remain  fluid.  In  litmus  milk  our  organism  produces  very 
slight  acidity  after  some  days.  In  this  respect  it  seems  to  differ 
slightly  from  those  as  described  by  others,  which  become  markedly 
acid  in  twenty-four  hours. 

We  have  compared  our  organism  with  that  of  Howard  and 
Perkins,  obtained  through  the  kindness  of  Dr.  Libman,  of  Mt. 
Sinai  Hospital,  New  York.  In  morphology  and  character  of 
growth  on  blood-agar  and  in  dextrose-agar  they  are  identical. 
They  react  in  the  senim-water-sugar  media  in  much  the  same 
way.  Both  coagulate  the  sugar-containing  media  before  men- 
tioned, but  there  are  slight  differences  in  the  reaction  time.     To 


92 


Studies  on  the  Pneumococcus 


compare  the  acid  production  we  have  added  0.5  c.c.  of  fresh 
sterile  defibrinated  horse's  blood  to  tubes  containing  10  c.c.  of 
bomllon  with  i  %  of  the  various  sugars.  The  results  are  shown 
in  the  following  table : 


•J 

Acidity  in  Percentage  of  Normal 
Acid  Solution. 

Our 
Organism. 

Organism  of 

Howard  and 

Perkins. 

Bouillon  plus  blood 

1-1% 
1.6% 
1.6% 
4.0% 

4-0  % 
3-9% 
4.1% 
4-o% 

1-7  % 

Bouillon  plus  blood  with  i  %  mannit    

1-4% 

Bouillon  plus  blood  with  i  %  inulin 

3-4% 

Bouillon  plus  blood  with  i  %  dextrose 

4-0  % 

Bouillon  plus  blood  with  i  %  lactose 

3-8% 

Bouillon  plus  blood  with  i  %  maltose 

3-8% 

Bouillon  plus  blood  with  i  %  galactose 

4.0  % 

Bouillon  plus  blood  with  i  %  saccharose    

Bouillon  plus  blood  with  i  %  dextrin    

3-8% 

Uninoculated  control  .9  % 


The  figures  show  that  our  organism  differs  from  that  of  Howard 
and  Perkins  in  the  amount  of  acid  produced  in  the  presence  of 
inulin  under  given  conditions.  Neither  of  them  produces  marked 
acidity  in  the  mannit-blood-bouillon.  Since  the  serum-water 
medium  with  the  addition  of  blood  and  each  of  these  sugars  was 
coagulated,  the  greater  delicacy  of  the  titration  method  of  de- 
monstrating these  reactions  is  again  shown.  When  the  total 
acidity  produced  by  a  culture  under  the  conditions  of  the  ex- 
periment rests  at  about  1.5  %  normal  acid,  the  reaction  in  the 
serum- water  medium  is  apt  to  be  imcertain.  The  identity  of  the 
reaction  in  serum-water  with  that  in  bouillon  is  again  brought  in 
question. 

The  hacmolytic  activity  of  these  two  cultures  has  been  roughly 
estimated  in  the  following  way :  to  tubes  of  plain  beef  bouillon 
.5  c.c.  of  defibrinated  horse-blood  are  added.  Tubes  are  inocu- 
lated and  incubated  with  controls.  In  twenty-four  hours  the 
height  to  which  the  color  ring  of  dissolved  haemoglobin  rises  in  the 


Charles  W.  Duval  and  Paul  A.  Lewis  93 

tube  is  noted.  The  haemolysis  is  markedly  greater  with  our 
organism  than  with  that  of  Howard  and  Perkins.  This  seems  to 
agree  with  the  fact  that  when  our  culture  was  first  isolated  we 
had  great  difficulty  in  growing  it  except  in  the  presence  of  blood. 
No  such  difficulty  is  noted  by  others  who  have  isolated  the  or- 
ganism. Efforts  to  obtain  a  haemolysin  or  toxin  that  would  pass 
a  filter  have  so  far  been  without  result. 

Pathogenicity. — The  diplococcus  with  which  we  have  worked 
is  pathogenic  for  mice,  guinea-pigs,  and  rabbits.  When  first 
isolated,  one  loop  of  blood-agar  culture  twenty-four  hours  old 
sufficed  to  kill  a  guinea-pig  or  yotuig  rabbit  in  twenty-four  hours 
with  general  septicemia  and  local  exudation.  At  present  several 
loops  are  necessary  to  bring  about  the  same  result.  Smaller 
doses  cause  death  in  several  days  with  fibrinous  inflammations 
of  the  serous  cavities.  The  exudate  is  characteristic ;  with  some 
exceptions  the  amotrnt  of  fibrin  is  small,  but  the  total  exudate  is 
relatively  large.  It  is  white,  and  has  the  consistency  of  thick 
mucilage.  If  dropped  from  a  pipette  into  salt  solution,  the  drop 
holds  together,  sinks  to  the  bottom,  and  remains  intact  for  some 
time.  There  is  little  tendency  to  hsemorrhage  either  after  sub- 
cutaneous or  intraperitoneal  inoculation.  One  old  rabbit  which 
survived  a  small  dose  developed  after  several  weeks  multiple 
arthritis  with  much  exudate  in  the  joint  cavities. 

Previous  observers  have  considered  this  organism  a  strepto- 
coccus. The  names  Streptococcus  capsulatus  and  Streptococcus 
mucosus  have  been  applied  to  it.  Morphologically  it  seems  much 
more  closely  related  to  the  pneumococcus.  The  lanceolate 
diplococcus  form  and  arrangement  are  well  marked,  as  shown  by 
Fig.  I ,  and  are  well  preserved  under  prolonged  cultivation.  Chains 
occur,  but  they  are  no  larger  than  is  usual  with  the  pneumococcus. 
They  seem  to  occur  under  much  the  same  unfavorable  conditions 
that  tend  to  chain  formation  with  the  pneumococcus,  and  may 
possibly  represent  an  involution.  In  its  fermentation  reactions 
it  is  very  active  and  more  closely  resembles  the  pneumococcus 
than  the  streptococcus,  although  our  culture  differs  from  that  of 
Howard  and  Perkins  in  this  respect.  Its  pathological  reactions 
also  resemble  the  pneumococcus  in  that  the  organism  appears  in 


94  Studies  on  the  Pneumococcus 

the  blood  after  injection  of  pure  or  mixed  culture  somewhat  more 
readily  than  does  the  streptococcus.  The  organism  is  apparently 
a  highly  specialized  pneumococcus. 

DESCRIPTION  OF  PLATE. 

Fig.  I. — Streptococcus  mucosus.  Stained  by  Gram's  method  and  magnified 
looo  diameters  to  show  the  morphology  of  the  culture  described. 

Figs.  2,  3,  4. — Streptococcus  mucosus.  Stained  by  the  modified  Gram's 
method  and  magnified  1000  diameters  to  show  capsules. 

a.  Single  coccus  with  complete  capsule. 

b.  Capsule   shows   slight   lateral   thickening   and   line   projecting  toward 

center. 

c.  Very  heavy  lateral  thickenings  in  capsule. 

d.  Somewhat  heavier  lateral  thickenings  in  the  capsule. 

e.  Capsule  completely  surrounding  two  cocci. 

Fig.  5. — Pneumococcus  from  a  culture  on  blood-agar  twenty-four  hours  old. 
Stained  by  Hiss'  copper-sulphate  method  for  capsules  and  magnified  1000 
diameters.     No  serum  was  added  to  the  preparation. 

Fig.  6. — Culture  of  pneumococcus  (C.  D.  30)  on  blood-agar  twenty-four  hours 
old  (second  generation);  stained  by  Gram's  method  and  magnified  1000  diame- 
ters. Showing  the  formation  of  chains  composed  of  rather  distinct  lanceolate 
pairs. 


THE  JOURNAL  OF  EXPERIMENTAL  MEDICINE.    VOL.  Vll.  PLATE  XXIX. 


-  t    ,'  •     .      •      % 


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% 


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\  ^/ -   ^•^  :r : /  \ 


// 


vT. 


STUDIES     OF    THE     PNEUMOCOCCUS    AND     ALLIED 
ORGANISMS    WITH    REFERENCE    TO    THEIR    OC- 
CURRENCE IN  THE  HUMAN   MOUTH. 

By  LEO  BUERGER,  M.D. 
Assistant  in  the  Pathological  Laboratory,  Mt.  Sinai  Hospital,  New  York. 

The  work  upon  which  I  shall  report  in  this  paper  was  carried 
on  from  November,  1904,  to  April,  1905,  in  the  Laboratory  of 
the  Moimt  Sinai  Hospital,  at  the  request  of  the  Commission  for 
the  Investigation  of  Acute  Respiratory  Diseases,  of  the  New  York 
City  Board  of  Health. 

The  general  scope  is  set  forth  tm.der  the  following  headings : 
I. — The  occurrence  of  the  pneumococcus  in  the  mouths  of 

normal  individuals. 
II. — The  evidence  of  communicability  of  the  pnetimococcus 

from  one  person  to  another. 
III. — The  study  of  the  pneumococci  isolated. 
IV. — The  study  of  organisms  found  in  the  mouths  of  normal 
individuals  which  closely  resemble  the  pneumococcus. 
V. — The  identification  of  the  pneumococcus ;  features  of  most 

diagnostic  importance. 
VI. — The  agglutination  of  the  pneumococcus  and  alHed  organ- 
isms. 
VII. — General  summary. 

THE    OCCURRENCE  OF  THE  PNEUMOCOCCUS  IN  THE  MOUTHS  OF 
NORMAL   INDIVIDUALS. 

The  studies  on  the  occurrence  of  the  pneitmococcus  in  the 
mouths  of  normal  individuals  were  conducted  in  two  series. 

95 


96  Studies  of  the  Pneumococcus  and  Allied  Organisms 

1.  By  isolation  of  the  pneumococcus  from  the  mouths  of  ward 
patients,  doctors,  and  nurses,  not  suffering  from  pneumonia. 

2.  By  an  examination  of  throat  cultures  on  Loefiier's  serum 
taken  from  individuals  not  suffering  from  inflammations  with 
which  the  pneumococcus  is  usually  associated,  and  by  the 
morphological  identification  of  the  pneumococcus  in  such 
cultures. 

The  first  series  will  be  referred  to  as  the  ' '  completely  studied 
cases,"  the  second  as  the  "throat-culture  series." 

Completely  Studied  Cases. 

Technique. — The  material  from  which  the  isolations  were  made  was  obtained 
in  the  following  manner.  Sterilized  swabs,  such  as  are  used  for  making  throat 
cultures,  were  carried  to  the  bedside,  and  the  secretions  of  the  mouth  from  the 
region  of  the  posterior  pharyngeal  wall,  pillars  of  the  fauces,  and  tonsils  were 
obtained  by  means  of  them.  In  a  number  of  cases,  additional  material  for 
cultures  was  obtained  by  collecting  saliva  in  sterile  test-tubes. 

For  the  recognition  and  identification  of  the  pneumococcus  in  the  secretions, 
spreads  of  the  secretion  were  stained,  Loefiier  serum  cultures  were  examined, 
Petri  plates  were  prepared,  and  in  some  instances  animal  inoculations  were 
made. 

Very  little  reliance  was  placed  on  the  films.  In  most  instances  the  number 
of  organisms  present  in  the  original  material  was  so  small  that  they  were  not 
detected  in  the  films,  although  the  latter  were  stained  both  by  the  Gram  and 
the  capsule  methods.  At  first  thorough  and  painstaking  search  was  made  over 
these  specimens.  When  it  was  subsequently  found  that  the  examinations  were 
usually  negative,  even  when  the  culttire  method  proved  the  presence  of  the 
organism,  less  attention  was  paid  to  this  procedure. 

Loeffler  serum  tubes  were  inoculated  directly  from  the  swabs  in  every  case. 
The  tubes  were  incubated  and  spreads  from  various  parts  of  the  tube  were 
stained  after  from  eighteen  to  twenty-four  hours'  growth  by  the  author's  cap- 
sule method.  It  may  suffice  here  to  call  attention  to  the  fact  that  in  very  many, 
and  in  fact  in  the  majority  of,  instances,  the  presence  of  pneumococci  in  the 
mouth  can  be  diagnosticated  by  this  simple  procedure.  By  the  employment  of 
direct  inoculations  on  Loefiier  serum  in  every  case,  side  by  side  with  the  plate 
method,  I  was  able  to  determine  the  respective  values  of  these  two  procedures. 
The  full  data  will  be  given  under  the  section  dealing  with  the  incidence  of  the 
pneumococcus  in  normal  mouths. 

The  technique  employed  in  the  morphological  identification  of  the  organism 
on  the  LoefHer  serum  was  the  following:  In  most  instances  one  or  two  tubes 
were  inoculated  directly  by  means  of  swabs,  but  a  series  of  tubes,  the  inocula- 
tions being  made  on  successive  tubes  until  the  swab  was  fairly  dry,  was  alsa 
made  in  a  number  of  cases. 


Leo  Buerger  97 

The  plan  finally  adopted  was  to  examine  the  cultures  after  eighteen  to  twenty- 
four  hours'  growth,  the  spreads  being  made  as  follows:  A  number  of  loops 
from  various  parts  of  the  tube  was  removed  and  thoroughly  mixed  with  a  few 
drops  of  a  dilute  beef-serum  (beef-serum  i  and  normal  saline  solution  equal 
parts  by  volume)  previously  placed  upon  a  clean  slide.  A  loop  of  this  mixture 
was  carefully  spread  upon  a  perfectly  clean  cover-slip  and  stained  by  the 
author's  capsule  method.  2 

The  characteristic  appearance  of  the  pneumococcus  and  its  variations,  when 
stained  in  the  manner  described,  will  be  discussed  in  another  section.  The 
close  resemblance  of  this  organism  and  the  small  coccoid  and  diplococcoid 
forms  of  the  Friedlander  bacillus,  so  frequently  met  with  in  cultures  from  the 
human  mouth,  made  it  necessary  to  employ  some  differential  method.  The 
use  of  the  capsule  stain  combined  with  the  Gram  stain  made  the  differentiation 
of  the  two  organisms  in  spreads  possible.  It  was  found  that  both  the  capsules 
of  the  pneumococcus  and  Friedlander  bacillus  take  the  counterstain,  and  the 
body  of  the  former  remains  stained,  whereas  that  of  the  latter  is  decolorized. 

The  examination  of  the  Loefifler-serum  cultures  in  the  manner  described  was 
therefore  conducted  as  a  matter  of  routine  in  every  case.  It  served  as  a  fairly 
reliable  indicator  as  to  the  presence  of  pneumococci  in  the  plates.  The  value 
and  reliability  of  a  morphological  identification  of  the  organism  was  studied 
first,  by  comparing  the  results  obtained  in  the  plate  isolations  with  the  exami- 
nation of  the  throat  culture  in  every  case,  and  secondly  by  plating  out  a  large 
number  of  positive  tubes  and  demonstrating  the  presence  of  the  organism  in 
pure  cultvu-e. 

In  the  development  of  a  method  which  would  be  rapid  and  satisfactory  for 
the  isolation  of  the  pneumococcus,  a  number  of  factors  had  to  be  taken  into 
consideration.  It  was  deemed  important  in  dealing  with  an  organism  which 
is  known  to  suffer  such  considerable  alteration  in  morphology  and  virulence 
on  culture  media,  to  be  able  to  obtain  pure  cultures  which  would  be  but  little 
changed  from  the  original  type.  The  general  aim  was  to  observe  the  peculiari- 
ties of  morphology,  cultural  characteristics,  metabolic  activity,  and  virulence 
of  each  strain. 

The  two  methods  which  were  adopted  for  the  recovery  of  the  organism  were 

1  Undiluted  rich  pleural  or  ascitic  fluid  may  be  substituted. 

2  The  method  is  as  follows:  Before  the  spread  is  completely  dry  it  is  covered 
with  Zenker's  fluid  minus  acetic  acid  and  gently  warmed  for  3  to  5  seconds 
over  a  small  flame.  It  is  washed  rapidly  in  water  and  flushed  once  or  twice 
with  80  to  95  %  alcohol,  and  then  covered  with  tr.  iodine  which  is  allowed  to 
remain  on  the  cover-glass  from  30  to  60  seconds  or  even  longer.  The  iodine  is 
washed  off  in  alcohol  and  the  specimen  dried  in  the  air.  The  staining  is  done 
with  fresh  aniline  oil-gentian-violet  in  2  to  5  seconds,  the  excess  of  stain  being 
removed  with  2  %  salt  solution.  The  preparation  is  examined  in  this  fluid. 
See  the  Medical  News,  1904,  Ixxxviii,  11 17.  Double  staining  can  be  carried  out 
as  by  the  capsule  and  Gram  stains.  After  the  specimen  is  dried  in  the  air,  it  is 
stained  by  the  usual  Gram  method,  then  counterstained  with  strong  fuchsin 
(sat.  alcoholic  sol.  10  to  15  parts,  water  to  100  parts)  for  a  minute  or  longer, 
washed  in  water  and  mounted  in  that  fluid. 


98  Studies  of  the  Pneumococcus  and  Allied  Organisms 

first  plating  and  second  inoculation  of  animals.  The  former  is  more  tedious, 
the  latter  gives  one  a  strain  altered  perhaps  both  in  virulence  and  other  prop- 
erties. Dtu"ing  the  course  of  these  experiments  certain  modifications  and 
improvements  were  developed,  which  made  rae  adhere  to  the  plate  method 
throughout  the  series  of  experiments  to  which  I  have  referred. 

My  attention  was  directed  first  to  the  nature  of  the  medium  most  suitable 
for  plating,  and  second  to  the  plating  itself.  Observations  were  made  on  the 
rapidity  and  luxuriance  of  growth  of  a  number  of  pneumococci  on  serum-agar, 
and  on  serum-glucose-agar,  made  up  with  varying  amounts  of  peptone,  with 
or  without  the  use  of  meat  infusion,  and  of  various  titers.  Profuse  growths 
were  regularly  obtained  on  all  these  media. 

It  was  decided  to  employ  an  agar  or  a  2  %  glucose-agar  of  a  neutral,  or,  at 
most,  0.5  %  phenolphthalein  acid  titer.  The  medium  was  usually  made  from 
meat  infusion  and  contained  1.5  to  2  %  peptone  and  2.5  %  agar.  Stock  plates 
of  these  media  (serum-agar  and  2  %  glucose-serum-agar)  were  poured.  The 
agar  or  glucose-agar  was  melted  in  large  tubes  and  allowed  to  cool  down  to  a 
temperature  below  the  coagulation  point  of  the  serum.  One  third  volume  of 
rich  albuminous  ascitic  fitiid  was  added,  and  the  resulting  medium  poured  into 
Petri-plates.  These  were  tested  by  incubation  and  stored  in  the  ice-chest, 
ready  for  use. 

These  plates  were  used  for  isolating  the  pneumococcus.  At  first  the  serum- 
agar  and  glucose-serum-agar  were  used  side  by  side.  It  was  found  that  al- 
though the  former  gave  quite  as  luxuriant  growths  as  the  latter  on  slants,  for 
the  purposes  of  rapid  isolation  and  detection  of  the  organisms,  the  sugar  medium, 
as  pointed  out  by  Libman,^  was  to  be  preferred. 

Although  in  the  serum-glucose-agar  good  growths  are  obtainable  even  when 
the  reaction  of  the  original  agar  is  1.2  %  acid,  it  was  deemed  advisable  to  use  as 
low  a  titer  as  would  be  compatible  with  good  growth.  Neutral  glucose-agar, 
which  upon  the  addition  of  ascitic  fluid  became  slightly  alkaline,  afforded  a 
medium  which  was  very  favorable  for  the  growth  of  the  pneumococcus.  That 
the  acid  production  at  the  end  of  eighteen  to  twenty-four  hotirs  did  not  raise 
the  titer  of  such  a  medium  to  a  very  high  point  was  shown  by  employing  strepto- 
cocci which  were  known  to  be  abundant  acid  producers  in  glucose  media.  When 
cultivated  on  a  more  acid  glucose-serum-agar  (one  made  up  from  0.8  %  to  1.2  % 
acid  glucose-agar),  they  made  manifest  the  high  acid  titer  by  abundant 
precipitation  or  whitening  after  the  stated  period  of  time.  When  grown  on 
alkaline  agar,  this  was  not  apparent  until  almost  double  the  time  had 
elapsed. 

The  technique  of  plating  required  modification.  By  the  use  of  the  usual 
method,  very  many  colonies  are  buried,  and  as  deep  pneumococcus  colonies 
present  nothing  sufficiently  definite  to  aid  in  their  recognition  they  may  be 
overlooked.  When,  besides  this,  the  great  variety  of  the  flora  in  the  human 
mouth  and  the  usual  numerical  preponderance  of  other  organisms  over  the 
pneumococcus  are  considered,  it  is  clear  that  it  is  important  to  have  a  great 
number  of  surface  colonies  from  which  to  pick.  Both  the  usual  streak  method, 
and  one  carried  out  by  wiping  a  loop  over  the  whole  surface  of  a  plate,  by  a  to- 

3  Journal  of  Medical  Research,  1901,  vi,  54. 


Leo  Buerger  99 

and-fro  lateral  motion,  proved  unsuccessful.  The  plan  finally  adopted  was 
as  follows: 

A  swab  taken  from  the  mouth  was  thoroughly  shaken  in  a  tube  of  neutral 
bouillon.  From  this  primary  tube  dilutions  in  bouillon  with  four,  six,  and 
eight  loops  may  be  made.  A  small  portion  of  the  dilute  mixture  was  poured 
at  a  point  near  the  periphery  of  the  prepared  plates.  By  a  slight  tilting  motion 
the  fluid  was  carefully  distributed  over  the  whole  surface  of  the  plates.  Care 
must  be  taken  to  avoid  an  excess  of  fluid.  It  was  found  that  plates  made  in 
this  way  gave  a  sufficiently  thick  and  discrete  distribution  of  surface  colonies. 
As  a  rule,  a  number  of  plates  were  poured  from  the  eight-loop  mixture.  These 
were  generally  satisfactory.  In  plating  from  growths  on  Loeffler  serum  the 
same  plan  was  followed.  One,  two,  and  four  loop-full  mixtures  were  made 
from  a  primary  tube  containing  approximately  i^^th  loop  of  the  culture,  and 
poured  as  above. 

Films,  Loeffler  tubes,  and  plates  were  made  from  each  case.  Eighteen  to 
twenty- four  hours  later  the  examinations  of  the  cultures  were  made.  As 
already  stated,  the  spreads  were  usually  negative,  while  the  Loeffler  cultures 
often  showed  the  presence  of  pneumococci.  The  plates  were  also  studied  for 
the  characteristic  colonies. 

The  colonies  of  pneumococci  are  described  rather  indefinitely  by  most  authors 
as  being  colorless,  almost  transparent,  and  watery.  When  studied  on  the 
media  recommended,  many  strains  present  very  characteristic  colonies.  The 
surface  colonies  of  the  pneumococcus  take  on  the  form  of  small  flattened  disc- 
like growths.  When  viewed  from  above,  and  by  means  of  reflected  light,  the 
surface  looks  glassy,  either  flat  or  slightly  depressed  in  the  center.  When 
looked  at  from  the  side,  or  more  especially  by  transmitted  light,  the  colonies 
appear  as  distinct  rings,  enclosing  an  almost  transparent  central  area.  When 
held  up  against  the  light,  these  rings  look  milky  and  somewhat  opaque.  The 
ring  form  is  very  characteristic  for  most  of  the  pneumococci  directly  isolated 
from  the  mouth.  It  must  not  be  understood  that  all  pneumococci  grow  in  this 
way,  or  that  the  same  organism  will  do  so  under  all  conditions.  At  times  a 
more  mucoid  colony  without  the  ring  shape  is  the  type  for  some  strains.  How- 
ever, a  sufficiently  large  number  of  the  organisms  were  found  to  show  this  form 
to  warrant  a  careful  search  for  them  in  the  plates.  Streptococci  were  found  in 
rare  instances  to  grow  in  ring-like  colonies.  These,  however,  when  carefully 
examined  were  found  to  have  a  distinct  nucleus,  or  more  opaque  center,  thus 
differing  from  the  transparent  almost  invisible  center  of  the  pneumococcus 
colonies. 

It  was  customary  to  pick  off  a  portion  of  the  suspected  colonies  and  stain 
it  by  means  of  the  capsule  method.  When  the  typical  ''ring- form"  was 
present,  the  typical  encapsulated  pneumococcus  forms  were  generally  found. 
When  the  colonies  were  too  small,  subcultures  were  directly  made,  and  the 
morphological  identification  postponed  a  day.  Positive  colonies,  spreads  of 
which  had  shown  encapsulated  diplococci,  were  cultivated  on  tubes  of  serum- 
agar.  From  this  point  on,  sugar  media  were  avoided,  and  transplantations 
were  regularly  made  on  ascitic-serum-agar.  Twenty-four  hours  later  the  sur- 
face growth  was  employed  for  tests  of  pathogenicity,  and  subcultures  used  for 
further  morphological,  cultural,  and  other  tests.     In  this  way  it  was  possible 


100  Studies  of  the  Pneumococcus  and  Allied  Organisms 

in  most  instances  to  inoculate  test  animals  with  pure  cultures  of  the  organism 
forty-eight  hours  after  the  plates  were  poured,  and  with  the  second  generation 
of  the  organism. 

White  mice  were  employed  for  tests  of  virulence,  the  heightening  of  the 
virulence  of  certain  strains,  for  testing  doubtful  organisms,  and  for  direct 
inoculations  of  saliva. 

In  testing  virulence,  inoculation,  whenever  possible,  was  made  with  the 
second  generation  of  the  pneumococci.  The  surface  growth  of  a  24-hour  culture 
on  serum-agar  was  suspended  in  i  c.c.  of  normal  saline  solution  and  injected 
subcutaneously.  These  animals  served  not  only  as  a  means  for  determining 
the  pathogenicity  of  the  organisms,  but  also  to  prove  the  correctness  of  the 
diagnosis  by  presenting  the  typical  encapsulated  forms  in  the  exudate  and 
blood. 

A  heightening  of  virulence  was  resorted  to  for  the  purpose  of  obtaining  strains 
which  were  to  be  used  for  the  immunization  of  rabbits.  Tests  to  increase  the 
virulence  of  organisms  resembling  the  pneumococcus,  and  of  avirulent  forms 
of  the  pneumococcus  were  also  made. 

Doubtful  organisms,  particularly  those  in  which  typical  capsule  forms  were 
not  demonstrable  in  culture,  were  also  put  into  mice  for  purposes  of  diagnosis. 


CONSIDERATION    OF   THE    CASES. 

In  this  place  will  be  given  the  results  of  the  studies  on  the 
occurrence  of  the  pneumococcus  in  normal  mouths.  Although 
the  mouths  were  not  always  strictly  normal,  those  which  for  all 
intents  and  purposes  could  be  regarded  as  such,  fell  within  the 
scope  of  the  work.  In  some  cases  there  was  slight  hypersemia, 
moderate  catarrhal  inflammation,  or  slight  follicular  hyperplasia. 
Notes  on  these  conditions  were  always  made. 

The  material  for  study  was  derived  in  the  main  from  the  hos- 
pital wards.  The  total  number  of  normal  cases  studied  was  78. 
Cultures  were  also  taken  from  doctors,  nurses  on  duty,  and  a  few 
from  private  persons  outside  the  hospital.  Some  of  the  diagnoses 
in  the  sick  were  multiple  neuritis,  brain  tumor,  rheumatism, 
osteomyelitis,  appendicitis,  nephritis,  and  cirrhosis  of  the  liver. 
In  a  general  way  it  may  be  said  that  the  work  was  planned  with 
a  view  of  obtaining  evidence  of  communicability  from  person  to 
person  of  the  pneumococcus.  Negative  cases  were  examined 
two,  three,  or  more  times  in  the  attempt  to  discover  the  organism, 
and  positive  cases  were  also  re-examined.  In  the  latter,  data 
regarding  the  persistence  of  pneumococci  in  the  mouth  were 


Leo  Buerger 


101 


sought.  The  78  persons  studied  consisted  of  49  adult  males, 
24  adult  females,  and  5  children. 

The  percentage  of  those  in  which  pneumococci  were  found  at 
some  time  or  other  was  fifty. 

The  following  tabulation  gives  the  percentage  of  positive 
results  in  the  case  of  adult  males,  females,  and  children. 


Total  Number. 

Positive. 

Percentage. 

Adult  Males 

49 
24 

5 

25 
1 1 

3 

=;i  .0 

Adult  Females 

45-8 

Children 

60.0 

In  the  above  so-called  "normal  cases"  there  was  one  child  in 
whom  diphtheria  bacilli  were  isolated  from  the  throat. 

In  addition  to  the  normal  cases,  the  presence  of  pneumococci 
was  determined  also  in  fifteen  cases  of  pneumonia.  Most  of  these 
were  cases  of  frank  lobar  pneumonia,  others  were  cases  of  broncho- 
pneumonia, one  was  a  case  of  grippe-pneumonia,  while  in  one 
case  only  was  the  Friedlander  bacillus  found.  These  patho- 
logical cases  were  added  to  the  list  for  the  following  reasons: 
first  in  order  to  obtain  information  regarding  the  reliability  of 
the  cultural  methods  employed;  second  as  controls  in  the 
studies  made  upon  the  communicability  of  the  pneumococcus 
from  the  mouths  of  pneumonia  cases  to  other  patients  in  the 
ward ;  and  third  for  a  comparative  study  of  the  organisms  iso- 
lated from  them  and  from  normal  persons. 

Of  the  total  fifteen  cases  there  was  one  case  as  stated  in  which 
the  Friedlander  bacillus  but  not  the  pneumococcus  was  isolated, 
and  another  case  of  "influenza  pneumonia"  in  which  neither 
pneumococcus  nor  the  pneumo-bacillus  could  be  discovered. 
In  the  remaining  thirteen  cases  the  Diplococcus  lanceolatus  was 
demonstrated  once  or  more  times.  Twelve  strains  were  isolated 
and  studied.  By  repeated  examinations  of  the  cases,  information 
regarding  the  persistence  of  the  organism  in  the  mouths  of  such 
patients  was  obtained. 

Before  entering  upon  a  more  detailed  study  of  the  normal  and 


102 


Studies  of  the  Pneumococcus  and  Allied  OrganisT/is 


pnetunonia  cases,  it  may  be  well  to  append  a  table  (I) 
the  condition  of  mouth  and  pharynx  in  each  patient. 


givmg 


TABLE  L 

CONDITION    OF 

THE    MOUTH    AND    PHARYNX. 

Case 

Case 

Case 

No. 

No. 

No. 

I. 

Follicular  hypertrophy. 

32. 

Negative. 

63. 

Negative. 

2. 

Negative. 

33- 

** 

64. 

"     (pneumonia) 

3- 

' '            (pneumonia) 

•  34- 

"     (pneumonia). 

65- 

"                " 

4- 

Diphtheria. 

35- 

Negative. 

66. 

"                " 

5- 

Large  left  tonsil. 

36. 

Slight  pharyngitis. 

67. 

Negative. 

6. 

Negative. 

37- 

Neg.  (pneumonia). 

68. 

"     (pneumonia) 

7- 

" 

38. 

Negative. 

69. 

Negative. 

8. 

" 

39- 

Slight  congestion. 

70. 

" 

9- 

" 

40. 

Negative. 

71- 

" 

lO. 

Slight  pharyngitis. 

41. 

" 

72. 

" 

II. 

Negative. 

42. 

" 

73- 

" 

12. 

Slight  tonsillitis. 

43- 

" 

74- 

" 

13- 

Hyperaemia. 

44. 

" 

7S- 

" 

14. 

" 

45- 

"    (pneumonia). 

76. 

" 

IS- 

Negative. 

46. 

Slight  congestion. 

77- 

" 

16. 

" 

47- 

Negative. 

78. 

" 

17- 

" 

48. 

Slight  congestion. 

79- 

" 

18. 

" 

49- 

Negative. 

80. 

Congestion. 

19. 

' '     (pneumonia) . 

50- 

Slight  congestion. 

81. 

" 

20. 

Negative. 

SI- 

Negative. 

82. 

Occasional  congest. 

21. 

" 

52. 

" 

83- 

Large  tonsils. 

22. 

" 

53- 

" 

84. 

Congested  ? 

23- 

" 

54- 

Large  phar.  follicles. 

85. 

Slight  congestion. 

24. 

" 

SS- 

Negative. 

86. 

Negative. 

25- 

"     (pneumonia). 

56. 

" 

87. 

" 

26. 

Negative. 

57- 

" 

88. 

Congestion. 

27. 

" 

S8. 

" 

89. 

" 

28. 

"     (pneumonia). 

59- 

" 

90. 

Negative. 

29. 

Negative. 

60. 

"     (pneumonia). 

91. 

" 

3°- 

" 

61. 

"              " 

92. 

Congestion. 

31- 

"     (pneumonia). 

62. 

((              (i 

93- 

Negative. 

A  survey  of  this  table  will  show  that,  apart  from  slight  con- 
gestion (which  was  not  present  at  every  examination)  or  folHcu- 
lar  hyperplasia,  most  of  the  throats  examined  were  negative 
for  pathological  conditions.  The  cases  are  given  in  the  order 
of  their  study. 

In  order  to  facilitate  future  reference,  both  as  to  the  organisms 
isolated  and  the  cases  themselves,  I  have  tabulated  the  com- 
pletely studied  cases,  indicating  whether  they  were  normal 
or  pneumonia  cases,  and  also  noting  the  number  of  the  cultures 
of  pneimiococcus  or  allied  organisms.  Whenever  more  than 
one  culture  was  isolated  for  thorough  study,  more  than  one 
number  will  appear  in  the  respective  column. 


Leo  Buerger 
TABLE  II. 

CASES    AND    CULTURES. 


103 


Case. 

Name. 

Pneumococcus** 

Not  Studied,  t 

Other  Organ- 
isms, t 

I. 

H.  S. 

N* 

2. 

J-F- 

N 

— 

+ 

— 

3- 

S.  R. 

P 

E  I 

— 

— 

4- 

A.  L. 

N 

E  2 

— 

— 

5- 

S.  S. 

N 

— 

— 

— 

6. 

A.  B. 

N 

— 

— 

— 

7- 

M.  F. 

N 

— 

— 

— 

8. 

G.  S. 

N 

— 

— 

— 

9- 

J.  E. 

N 

— 

— 

— 

lO. 

L.  B. 

N 

— 

— 

— 

II. 

I.  H. 

N 

E3 

— 

— 

12. 

Private  E.  L. 

N 

E4 

— 

— 

13- 

A.  H. 

N 

Es 

— 

— 

14. 

Private  E.  Z. 

N 

E6 

— 

— 

IS- 

J.  H. 

N 

— 

— 

— 

16. 

S.  K. 

N 

— 

— 

— 

17- 

Nurse  S. 

N 

— 

— 

— 

18. 

S.  G. 

N 

— 

— 

— 

19. 

S.  S. 

P 

E7 

— 

— 

20. 

B.  R. 

N 

— 

— 

21. 

A.  St. 

N 

— 

— 

— 

22. 

Nurse  G. 

N 

— 

— 

E31 

23. 

Nurse  M. 

N 

— 

+ 

24. 

S.  Sh. 

N 

E  16,  19 

— 

— 

25- 

A.  Se. 

P 

— 

— 

F 

26. 

Al.  B. 

N 

— 

— 

— 

27. 

Nurse  W. 

N 

— 

— 

— 

28. 

L.  G. 

P 

E8 

— 

— 

29. 

W.  B. 

N 

E  9 

— 

— 

3°- 

J.  M. 

N 

— 

+ 

— 

31- 

H.  C. 

P 

E  10 

— 

— 

32. 

S.  St. 

N 

E  II 

— 

— 

33- 

N.  G. 

N 

E  12 

— 

— 

34- 

M.  S. 

P 

E13 

— 

— 

35- 

H.  K. 

N 

E  14 

— 

— 

36. 

N.  S. 

N 

E  15,  20,  22 

— 

— 

37- 

T.  C. 

P 

— 

+ 

— 

38. 

J.  B. 

N 

— 

+ 

E  42 

39- 

H.  P. 

N 

E  18 

— 

— 

40. 

E.  W. 

N 

— 

— 

F 

41. 

A.  B. 

N 

E  17 

— 

— 

42. 

S.  W. 

N 

E  24 

— 

— 

*  N  means  "normal";   P,  pneumonia;  F,  Friedlander  bacillus  infection. 

**  Cultures  in  this  column  were  identified  as  pneumococci  and  will  be  desig- 
nated by  the  letter  E  followed  by  a  number.  They  were  isolated  from  the  case 
occupying  the  same  line. 

t  The  plus  mark  indicates  that  in  these  cases  pneumococci  were  found  but  not 
completely  studied. 

t  Other  organisms,  i.  e.,  doubtful  strains,  unidentified  forms,  and  Strepto- 
coccus mucosus  capsulatus  are  designated  by  letter  E  or  otherwise. 


104  Studies  of  the  Pneumococcus  and  Allied  Organisms 

TABLE  II  {Continued). 


Case 

Name. 

Pneumococcus. 

Not  Studied. 

Other  Organ- 
isms. 

43 

M.  S. 

N 

E  21 





44 

M.  St. 

N 

E  48 

+  § 

— 

45 

B.  R. 

P 

E23 

— 

— 

46 

B.  S. 

N 

E  40 

+ 

— 

47 

S.  C. 

N 

— 

— 

— 

48 

I.  P. 

N 

— 

— 

— 

49 

M   So. 

■  N 

— 

— 

— 

50 

M.  G. 

N 

— 

— 

F 

51 

R.  Kr. 

N 

E25 

— 

— ■ 

52 

A.  Be. 

N 

— 

— 

53 

H.  W. 

N 

E  26 

— 

— 

54 

H.  Ka. 

N 

E  27 

— 

+  § 

55 

A.  Kr. 

N 

E39 

— 

E30 

56 

T.  S. 

N 

— 

— 

57 

N.  M. 

N 

E  28,  29 

— 

— 

58 

A.  S. 

N 

— 

+ 

— 

59 

M.  W. 

N 

— 

— 

— 

60 

D.  B. 

P 

E32 

— 

— 

61 

F.  T. 

P 

E  Z3^  36,  37.  38 

— 

— 

62. 

M.  G.  1 

P 

— 

— 

63 

S.  K. 

N 

E45 

— 

E  41 

64. 

R.  M. 

P 

— 

+ 

— 

65- 

A.  S. 

P 

E35 

— 

— 

66 

M.  Bo. 

P 

E34 

— 

— 

67 

I.  K. 

N 

— 

— 

68 

M.  Rei. 

P 

E43 

— 

— 

69 

F.  C. 

N 

E  47.  54 

— 

— 

70 

A.  C. 

N 

E  44,  46 

— 

— 

71 

F.  R. 

N 

— 

+ 

— 

72 

L.  G. 

N 

— 

+ 

— 

73 

R.  E. 

N 

— 

— 

— 

74 

D.  G. 

N 

— 

— 

— 

75 

S.  F. 

N 

— 

— 

— 

76 

H.  G. 

N 

E49 

— 

— 

77 

E.  M. 

N 

— 

— 

78 

K.J. 

N 

— 

— 

— 

79 

L.  W. 

N 

— 

— 

— 

80 

A.  N. 

N 

E  so 

— 

— 

81 

H.  C. 

N 

E51 

— 

— 

82 

M.  Fe. 

N 

— 

— 

83 

I.  B. 

N 

— 

— 

— 

84 

E.  A. 

N 

— 

— 

— 

85 

A.  Bo. 

N 

E52,  56 

— 

— 

86 

C.  W. 

N 

E53 

— 

— 

87 

.       J.  J- 

N 

— 

— 

88 

B.  St. 

N 

— 

— 

— 

89 

.       N.  H. 

N 

— 

— ^ 

— 

90 

L.  W. 

N 

— 

— 

— 

91 

.       B.  H. 

N 

— 

+ 

E57 

92 

.       M.  St. 

N 

— 

— 

— 

93 

.       B.  Ko. 

N 

— 

E55 

S.  M.t 

§  In  these  cases  at  one  time  the  organism  was  identified  but  not  studied ;  at 
another  time  culture  marked  E  was  isolated. 

II  Influenza  pneumonia  case.  IT  Streptococcus  mucosus. 


Leo  Buerger  105 

The  Persistence  of  the  Pneumococcus  in  the  Mouths  of  Normal 
Persons. — In  the  study  of  the  organism  in  the  so-called  normal 
cases,  the  repeated  occurrence  of  the  pneumococcus  in  the 
same  individual  was  often  demonstrated.  Time  did  not  per- 
mit of  the  complete  study  of  all  the  organisms  isolated  in 
this  way.  Whenever  the  persons  themselves  or  the  particular 
strain  of  organisms  presented  points  of  unusual  interest,  a  ntim- 
ber  of  different  strains  was  isolated  and  studied.  Reference  to 
the  foregoing  table  will  show  that  in  some  patients,  two,  three, 
or  four  pneumococci  (marked  E)  obtained  on  different  occasions 
were  identified  and  kept  for  study.  In  many  of  the  other  cases, 
however,  when  cultures  were  repeatedly  positive  the  fact  alone 
was  recorded,  and  the  organism  itself  merely  identified. 

For  the  purpose  of  convenience  the  results  of  the  observations 
on  a  number  of  the  cases  in  which  the  pneumococcus  was  found 
more  than  once  are  tabulated.  A  number  of  instances  in  which 
there  were  several  negative  cultures  and  one  positive  one  is 
also  included.  The  data  may  be  taken  as  showing  that  the 
pneimiococcus  is  to  be  found  in  normal  human  mouths  for  days, 
weeks,  or  even  longer. 

In  the  following  table  the  words  "positive"  and  "negative"  * 
indicate  whether  or  not  pneumococci  were  cultivated  from  the 
mouth  on  the  corresponding  dates : 

TABLE  III. 

PNEUMOCOCCUS  IN  THE  NORMAL  MOUTH. 

Case  2.  Case  24.  Case  2g. 

Nov.  25,  negative.                  Dec.     4,  negative.  Dec.     7,  negative. 

"     27,  positive.                       "  10,        '_'  _  "  18^  positive. 

Dec.     4,  negative.                      "  16,  positive.  "  26,  negative. 

"     II,        "                               "  19,  negative.  "  ,j' 

"     20,       "                               "  20,  positive.  I^n.  20         " 

"     28,       "  "  23,  negative. 

"  27,  positive. 

Case  13.  "  28,        "    _  Case  30. 

Jan.      I,  negative. 

Dec.     3,  positive.                       "       3,  positive.  Dec.  20,  negative. 

"       9,        "                               "       4,        "  "23,  positive. 

"     18,       "                               "       6,  negative.  "  27,  negative. 

■»  The  phrases  "positive  case"  and  " negative  case "  are  used  throughout  this 
paper  to  indicate  the  presence  or  absence  of  pneumococci  in  the  mouth. 


106  Studies  of  the  Pneuraococcus  and  Allied  Organisms 


Dec. 


Dec. 


Dec. 
Jan. 


Case  J2. 

20,  positive. 

21,  " 
26, 

31,  negative. 

Case  35. 
20,  positive. 
23. 

Case  j6. 

27,  positive. 
28, 

I, 

6, 

7. 


Jan. 


Feb. 


Jan. 


Feb. 


Feb. 


Feb. 


Feb. 
Mar. 


Case  57. 

19,  positive. 

20,  " 

21,  " 

5,  negative. 

Case  6 J. 

24,  negative. 
26, 

29,  positive. 
7,  negative. 


2, 

13. 
28, 


Case  6g. 
,  positive. 


TABLE  III  {Continued). 
Case  42 
Dec.  27,  negative. 
Jan.      4,  positive. 

8,  negative. 

9,  positive. 
II,  negative. 

18,  positive. 

24,  negative. 

Case  51. 

Jan.    II,  negative. 

"      14,  positive. 

"      16, 

"      18,  negative. 
"     24, 
Feb.     7, 

Case  54. 
Jan.    14,  positive. 
!!     IS.        ]\ 

^^^^  39-  '    Case  55- 

Dec.  28,  positive.  Jan.  11,  negative. 

"     29,        "  "  IS, 

"     30.       "    .  "  16, 

Jan.     3,  negative.  "  19,       " 

"      16,  positive.  "  26,  positive. 

"     19.        "    .  "  27,       "    _ 

"     29,  negative.  "  29,  negative. 

Before  analyzing  this  table  I  wish  to  present  the  results  of  the 
examinations  in  another  form.  In  this  the  longest  time  which 
elapsed  between  two  positive  cultures  in  the  same  case  will  be 
indicated,  and  the  total  number  of  examinations  made  in  each 
instance  and  the  total  number  of  positive  findings  recorded.  All 
the  cases  in  the  last  table  are  not  included  in  Table  IV, 


Case  JO. 
,    positive. 

Case  85. 


23,  positive. 
2,       " 


TABLE 

IV. 

Case. 

Time.* 

Examinations,  t 

No.  Positive. J 

13 

15 

3 

3 

24 

19 

II 

6 

32 

6 

4 

3 

35 

4 

3 

3 

36 

12 

6 

6 

39 

22 

7 

S 

42 

14 

8 

3 

69 

26 

3 

3 

70 

4 

2 

2 

85 

10 

2 

2 

Total 

10 

— 

49 

36 

*  The  number  of  days  which  elapsed  between  the  first  and  last  examination. 
t  The  number  of  examinations  made  on  different  days. 
X  The  number  of  times  pneumococci  were  found. 

Cases  in  which  only  one  positive  culture  was  obtained  and  those  in  which  the 
time  between  two  positive  examinations  was  only  one  or  two  days  are  omitted. 


Leo  Buerger  107 

Examination  of  Tables  III  and  IV  brings  out  some  interesting 
points.  In  the  first  place  it  is  seen  that  one  or  more  negative 
examinations  often  preceded  the  positive  finding.  A  single  set 
of  negative  cultures  is  of  course  not  convincing.  In  some  cases, 
however,  the  absence  of  the  organism  was  noted  on  two,  three, 
or  more  days  before  the  first  positive  find.  Such  cases  are 
certainly  to  be  regarded  as  furnishing  evidence  of  the  fact  that 
the  pneumococcus  may  suddenly  appear  in  normal  mouths 
which  have  been  previously  shown  to  be  free  from  them.  The 
following  are  some  such  instances : 

Case    2,  negative,  Nov.  25;  positive,  Nov.  27. 

"    55,  negative,  Jan.  11,  15,  16,  19;       positive,  Jan.  26. 
"    63,  negative,  Jan.  24,  26,  27;  positive,  Jan.  29. 

Another  fact  to  be  mentioned  is  the  regularity  with  which  the 
pneumococcus  was  found  in  certain  persons.  Thus  it  was 
present : 

In  Case  13,  3  times  in  3  examinations. 

"  "  35.  3  "  "3 
"  "  36,  6  "  "6 
"       "     69,  3       "      "3 

In  the  latter,  the  intervals  between  the  first  and  last  examina- 
tion were  15,  4,  12,  and  26  days,  respectively.  These  patients  of 
this  table  left  the  hospital  shortly  after  the  last  culture  was 
taken  and  hence  could  not  be  studied  further. 

The  second  column  of  Table  IV  shows  how  long  pneumococci 
were  actually  found  to  exist  in  some  of  the  cases.  The  shortest 
time  is  four  days,  the  longest  twenty-six.  There  seems  little 
doubt  that  these  figures  might  have  been  larger  had  the  ob- 
servations been  extended  over  a  longer  period  of  time. 

There  is  another  series  of  cases,  of  which  numbers  24,  39,  and 
42  are  examples  (Table  III),  which  deserves  mention.  In  these 
the  organism  was  not  detected  at  every  examination,  but  very 
frequently.  That  the  bacterial  flora  of  the  mouth  must  undergo 
changes  throughout  the  day  is  highly  probable.  The  mechanical 
and  chemical  influences  to  which  the  mucous  membrane  is 
subject  are  necessarily  great.     It  is  very  probable,  then,  that 


108 


Studies  of  the  Pneumococcus  and  Allied  Organisms 


repeated  examinations  made  on  the  same  day  might  have  re- 
vealed the  organism  where  a  single  ctdture  proved  negative. 

From  a  consideration  of  the  foregoing,  there  would  seem  to  be 
evidence  that  the  pneiimococcus  may  suddenly  appear  in  the 
so-called  normal  mouth,  that  certain  individuals  cany^  the  or- 
ganism in  their  mouths  for  considerable  periods  of  time,  and  that 
even  those  persons  in  whom  the  organism  cannot  be  demon- 
strated at  every  examination  may  harbor  the  pneumococcus  in 
the  less  accessible  portions  of  the  respiratory  tract. 

THE   STUDY   OF    CASES    OF    PNEUMONIA. 

From  this  class  of  patients,  as  already  mentioned,  the  pneu- 
mococcus was  isolated  as  a  control  in  the  study  of  the  communi- 
cability  of  the  pneumococcus  to  other  patients  in  the  hospital 
wards.  These  cases  also  furnished  proof  of  the  reliability  of  the 
cultural  methods  employed  and  materials  for  use  in  the  com- 
parative study  of  the  organisms  from  normal  and  pathological 
cases, 

TABLE  V. 

PNEUMONIA    CASES. 


Case. 

Pneumonia. 

Pneumococcus. 

3.  S.  R. 

Lobar 

positive. 

19,  J.  S. 

" 

25,  A.  S.* 

negative. 

28,  L.  G. 

positive. 

31,  H.  C. 

" 

34,  M.  S. 

" 

37,  T.  C. 

" 

45-  B.  R. 

" 

60,  D.  B. 

Broncho- 

" 

61,  F.  T. 

Lobar 

" 

62,  M.  G. 

Influenza 

negative. 

64,  R.  M. 

Lobar 

positive. 

65,  A.  S. 

" 

" 

66,  M.  B. 

Broncho- 

" 

68,  M.  R. 

Lobar 

With  the  exception  of  Nos.  25  and  62,  pneumococci  were  found  in  all.  The 
former  was  a  case  of  lobar  and  the  latter  a  case  of  influenzal  pneumonia.  There 
were  in  aU  12  lobar  and  two  broncho-pneumonic  cases. 

*  The  Friedlander  bacillus  was  isolated  repeatedly;  pneumococcus  not  foundt 


Leo  Buerger  109 

TABLE  VI. 

REPEATED    EXAMINATIONS    IN    CASES    OF    PNEUMONIA. 

Case  J.  Case  28. 


Nov.  22,  positive    (7th  day)  * 
Dec.      I,         "  — 

"       5,  negative  — 

Case  19. 

Dec.     3,  positive   (sth  day) 
8,         "  — 

17.         "  .  — 

21,  negative  — 

26,  positive  — 

31,  negative  — 


Dec.  15,  positive  (nth  day) 

"  17.         "  — 

"  23,  negative        — 

"  26,  positive  — 

"  31,  negative         — 

Case  ji. 

Dec.  18,  positive    (7th  day) 

"  27.         "  — 

Jan.  3,         "  — 


Case  61. 


Jan.  21,  positive  (?) 
"  22,  "  — 
"     23,         "         — 


*  Day  of  the  disease.  Whenever  this  column  is  found  blank,  the  examination 
was  made  after  resolution  had  set  in  or  the  crisis  had  occurred. 

There  was  no  difficulty  in  isolating  the  organism  from  these 
patients.  In  most  instances  several  successive  positive  cultures 
were  secured.  No  complete  investigation  of  the  length  of  time 
the  pneumococcus  could  remain  in  the  mouths  of  the  patients 
was  made.  Such  data  as  were  obtained  showed  that  they  were 
present  for  many  days  after  the  crisis  or  after  the  beginning  of 
resolution. 

In  Case  3,  it  was  foimd  9  days  after  the  crisis. ^ 

<<  a    „Q        n         it  a        ^^         <'  "  '<  " 

2o,  1 1 

<<  <'     -r^  i(  il  <'  r,-,  "  "  "  " 

Although  the  last  recorded  culture  taken  in  Cases  3,19,  and  28 
were  negative  (Table  VI),  it  is  possible  that  observations  ex- 
tending over  a  longer  period  of  time  may  have  given  positive 
results, 

PNEUMOCOCCI    IN    A    SERIES    OF    CULTURES    FROM    THE    THROAT. 

It  is  intended  in  this  section  to  give  the  results  of  studies  made 
upon  a  separate  series  of  cases.     The  plan  of  this  part  of  the 

5  Beginning  resolution  or  decided  drop  of  temperature  towards  normal. 


110  Studies  of  the  Pneumococcus  and  Allied  Organisms 

work  was  to  obtain  data  regarding  the  incidence  of  the  pneumo- 
coccus in  a  large  number  of  normal  cases,  to  ascertain  the  value 
of  the  morphological  identification  of  the  pneumococcus  as  com- 
pared with  the  other  methods,  and  to  determine  in  how  far  this 
last  procedure  would  fall  behind  the  plate  method  as  a  means 
for  detecting  the  organism  in  the  mouth. 

Two  hundred  and  four  normal  cases,  not  including  the 
seventy-eight  "completely  studied"  cases,  were  examined.  In 
this  series  there  were  71  positive  and  133  negative  cases,  or 
34.8  %  of  positive  cases,  being  15.2  %  less  than  the  percentage 
which  was  found  in  the  "completely  studied"  cases.  In  order 
to  explain  this  discrepancy  it  may  be  well  to  analyze  the  factors 
which  may  be  responsible  for  the  difference:  ist,  the  cases 
themselves;  2d,  the  imperfection  of  the  method,  and  3d,  the 
morphological  identification  of  the  pneumococcus  in  Loeffler  cul- 
tures. The  difference  in  percentage  may  after  all  fall  within  the 
limits  of  this  larger  series  of  cases.  But  the  fact  that  the  ex- 
aminations were  made  only  once  in  each  case  could  also  account 
for  the  disparity. 

The  cultures  were  often  taken  on  one  tube  only.  A  possible 
overgrowth  by  other  organisms  must  be  considered.  This  is  to 
be  avoided  in  many  instances  by  the  inoculation  of  several  tubes 
in  series  with  the  same  swab.  Although  in  the  preparation  of 
films  from  the  tubes  care  was  taken  to  include  many  different 
portions  of  the  surface  growth,  and  although  a  number  of  speci- 
mens were  examined  in  each  instance,  it  is  likely  that  a  certain 
n-umber  of  pneumococci  may  have  been  missed.  Finally  the 
morphological  identification  of  the  pneumococcus  may  be  at 
fault. 

In  the  spreads  stained  by  the  capsule  method,  the  pneumo- 
coccus must  be  differentiated  from  a  number  of  other  encapsu- 
lated organisms:  from  the  encapsulated  streptococcus,  the 
Streptococcus  mucosus  capsulatus,  the  diplococcoid  forms  of  the 
Friedlander  bacillus,  the  diplococcus  forms  of  the  Micrococcus 
tetragenus,  the  double  cocco-bacillary  organism  frequently 
found  in  beef -serum,  and  from  a  large  number  of  peculiar  en- 
capsulated diplococci  and  streptococci. 


Leo  Buerger  111 

In  145  of  the  204  throat  examinations,  notes  were  made  in 
regard  to  the  preponderating  organism.  In  the  following  table 
the  frequency  of  occurrence  of  the  organisms  appearing  in 
greatest  numbers  is  given  for  the  145  cases. 

TABLE  VII. 

PREPONDERATING    ORGANISMS    IN    THROAT    CULTURES. 

Streptococci 63 

Staphylococci 42 

Bacilli  (not  identified) 23 

Friedlander  bacilli 17 

In  this  series  the  Friedlander  bacillus  occurred  twenty-one 
times,  or  in  about  14.5  %,  and  the  Streptococcus  mucosus  cap- 
sulatus  eight  times,  or  in  about  5.5  %. 

In  many  of  the  cultures  the  mixture  of  organisms  was  so  great 
that  no  single  form  could  be  estimated  accurately.  Very  often 
the  streptococci  and  staphylococci  seemed  to  be  present  in 
equally  large  numbers.  Only  such  bacilli  were  identified  as 
belonging  to  the  Friedlander  group  as  exhibited  typical  cap- 
sules when  grown  on  Loeffler's  medium,  as  well  as  the  character- 
istic mucoid  growth.  There  was  an  exception  in  the  case  of  an 
organism  which  was  proven  to  belong  to  this  class  by  isolation 
and  subculture,  but  on  which  capsules  were  not  demonstrated. 
Many  of  the  bacilli  were  isolated  and  studied.  The  results  of 
this  part  of  the  study  furnish,  therefore,  further  evidence  of  the 
frequent  occurrence  of  the  pneumococcus  in  the  mouths  of  nor- 
mal individuals. 

STUDIES     ON     THE     COMMUNICABILITY     OF    THE     PNEUMOCOCCUS. 

This  part  of  our  study  was  approached  in  two  ways:  by 
means  of  the  histories  of  the  individuals  studied,  and  by  repeated 
observations  on  the  occurrence  of  the  pneumococcus  in  the 
mouths  of  patients  in  the  hospital  wards. 

Very  little  information  in  regard  to  the  source  of  the  pneumococci  to  be 
found  in  the  positive  cases  could  be  obtained  from  the  histories.  The  ignorance 
of  the  patients,  their  failure  to  remember  the  condition  of  those  with  whom 
they  came  into  contact,  and  their  inability,  in  most  instances,  to  give  reliable 
answers  in  regard  to  their  association  with  pneumonia  cases  made  obtaining  any 


112  StvAies  of  the  Pmumococcv.s  and  Allied  OrgoMisms 

reliable  data  unsatisfactory.  In  a  very  few  cases  there  was  a  history  of  pneu- 
monia in  some  member  of  the  family,  or  in  the  individual  himself.  One  posi- 
tive so-called  normal  case  had  lived  in  the  same  house  with  a  patient  who  was 
suffering  from  lobar  pneumonia,  and  who  was  subsequently  treated  in  the  same 
ward. 

Evidences  of  communication  of  pneiimococci  from  one  person 
to  another  were  sought :  by  demonstrating  the  absence  of  pneu- 
mococci  in  the  mouths  of  normal  cases,  by  noting  the  appearance 
of  the  organism  in  the  mouths  of  such  cases,  by  studying  the 
possible  sources  of  infection  and  groups  of  patients  who  came  in 
contact  either  with  pneumonia  cases  or  with  persons  in  whose 
mouths  the  pneumococcus  had  been  found,  and  by  collecting 
other  data  in  regard  to  the  occurrence  of  the  organism  elsewhere 
in  the  hospital  wards. 

For  this  study  two  male  wards,  one  containing  twenty-four 
and  the  other  twelve  beds,  and  a  medical  children's  ward  were 
selected.  The  ntunber  of  positive  cases  present  in  the  small 
ward  at  anv  given  time  was  rapidly  ascertained ;  three  successive 
days  sufficed  for  taking  cultures  from  all  twelve  cases,  four  being 
taken  at  a  time.  It  was  possible  by  the  employment  of  the 
plate  method  to  arrive  at  fairly  definite  conclusions  after  twenty- 
four  to  forty-eight  hours.  This  process  was  repeated  a  number 
of  times  and  the  subsequent  appearance  of  the  organism  in 
negative  cases  determined.  The  larger  male  ward  and  children's 
ward  were  studied  in  a  similar  manner,  five  or  six  ctiltures  being 
taken  a  day. 

Negative  cases  were  repeatedly  examined.  Some  of  them 
could  be  foUowed  from  the  day  of  their  arrival  in  the  wards. 
Others,  such  as  cases  of  chronic  nephritis,  cirrhosis  of  the  liver, 
chronic  rheumatism,  etc.,  had  already  been  occupants  of  beds 
for  davs  and  weeks  before  the  work  was  begun.  In  all  of  these 
it  was  possible  to  watch  for  subsequent  development  of  pneumo- 
cocci  in  the  mouth. 

The  group  of  cases  already  referred  to  was  selected  for  the 
purpose  of  observing  the  effect  of  the  association  of  "negative" 
individuals,  both  with  pneumonia  cases  and  with  those  "nor- 
mal"  persons   in  whom  large   numbers   of  pneumococci  were 


Leo  Buerger 


113 


repeatedly  found.  In  certain  circtimstances ,  upon  the  arrival  of 
a  pneumonia  patient  in  the  ward  the  secretions  from  the  mouth, 
as  well  as  from  the  mouths  of  patients  in  the  adjoining  beds, 
were  examined.  These  observations  were  repeated  on  a  numbeT 
of  days.  The  same  plan  was  adopted  for  the  study  of  some  of 
the  normal  "positive"  cases. 

In  regard  to  other  sources  of  infection,  a  number  of  handker- 
chiefs and  cups  belonging  to  "positive"  cases  were  examined  for 
the  presence  of  pneumococci. 

The  following  tables  will  give  the  results  of  several  series  of 
cases  studied  in  wards,  either  in  groups  or  singly.  Table  No. 
VIII  refers  to  one  of  the  children's  wards.  Isolated  observa- 
tions were  made  on  these  patients  during  a  period  of  about  one 
month  and  a  half. 

TABLE  VIII. 

OBSERVATIONS    ON    COMMUNICABILITY. 


Bed. 

Case. 

Nov.  22 

Dec.  4. 

6-io.t 

17-26 

Jan.  I. 

2-10. 

3-4. 

5-10. 

I 

2 

3 
5 
6 

7 
9 

normal  * 

neg. 

neg. 

— 

neg. 

pos. 
neg. 

pos. 

neg. 

neg. 

pneumonia 

normal 

pneumonia 

pos. 
pos. 

— 

pos. 
neg. 
pos. 

pos. 
neg. 

pos. 

— 

— 

neg. 
pos. 

neg. 

lO 

II 

12 

13 

NiiTse  I 
"      2 

normal 

— 

— 

neg. 

neg. 

— 

neg. 

— 

— 

pos. 
neg. 

pos. 

neg. 
pos. 

pos. 
pos. 

pos. 
neg. 

— 

neg. 

neg. 

— 

*  Normal,  i.  e.,  suflfering  from  some  disease  which  could  bear  no  relation  to 
the  presence  of  pneumococci  in  the  mouth. 

t  The  examinations  were  made  within  the  two  dates.  A  dash  signifies  that 
no  culture  was  taken;  usually  because  the  patient  had  left  the  ward  or  had 
not  as  yet  been  admitted. 

In  the  first  column  of  the  table  will  be  found  the  bed  num- 
bers of  the  cases  under  observation.  There  were  eleven  cases  in 
all.  Besides  these,  a  night  and  day  nurse  (Nos.  i  and  2)  are  in- 
cluded in  the  list.  On  November  2 2d  there  were  three  patients 
-with  pneumococci  in  their  mouths ;  two  of  these  were  cases  of 


114  Studies  of  the  Pneumococcus  and  Allied  Organisms 

pneumonia  and  one  a  "normal"  case.  On  the  4th  of  December 
Nurse  No.  2  was  positive,  but  inasmuch  as  she  had  not  been 
examined  before,  no  deduction  can  be  made.  On  the  17th  of 
December,  No.  9,  another  case  of  pneimionia,  was  admitted  to 
the  ward.  Bed  No.  i  now  furnished  an  instance  of  possible 
acquisition  of  the  organism  by  reason  of  association  with  positive 
cases.  A  girl  with  chorea  was  up  and  about  the  greater  part  of 
the  day  and  frequently  attended  to  the  wants  of  certain  other 
cases  in  the  ward.  Three  examinations  made  between  Novem- 
ber 2 2d  and  December  26th  gave  negative  results.  On  the  ist 
and  2d  of  January,  however,  the  organism  was  found  present  in 
her  mouth. 

As  an  example  of  a  case  in  which  the  appearance  of  pneimio- 
cocci  could  possibly  be  traced  to  the  patient  in  the  adjoining  bed, 
I  may  cite  the  following:  Case  24  (Table  II),  a  "normal" 
patient  in  Bed  i,  was  examined  on  the  4th  and  loth  of  December 
and  found  negative.  On  the  loth  of  December  a  case  of  lobar 
pneumonia  was  put  in  the  next  bed.  Pneumococci  were  re- 
peatedly demonstrated  in  the  latter  patient,  and  on  December 
1 5th  and  on  a  number  of  days  afterwards  the  same  organism  was 
detected  in  the  "normal"  patient,  as  is  shown  in  Table  IX. 

TABLE  IX. 


Bed  I. 

Bed  2. 

Bed4. 

Bed  I. 

Bed  2. 

Bed.  4. 

Dec.     4,  neg.* 
"     10,      " 
"     15,  pos. 
"     16,  pos. 
"     19.  neg. 
"     20,  pos. 
"     21      " 

pos. 
pos. 

Dec.  18,  pos. 

Dec.  23,  neg. 

"     27, t  pos. 

"     28,       " 
Jan.     I,  neg. 

"       3.  pos. 

"    neg. 

— 

pos. 
pos. 

In  the  above  table  the  dates  and  results  of  the  various  examinations  are 
given.  Another  case  of  lobar  pneumonia  (Bed  4)  was  admitted  on  December 
17th,  and  was  regularly  positive.  The  same  ward  furnished  cases  in  which 
pneumococci  were  only  occasionally  found.  The  source  of  the  organisms  could 
certainly  be  referred  to  the  large  number  of  patients,  pneumonia  or  normal, 
who  habitually  carried  them  in  their  mouths.  Thus  Bed  8,  in  Table  No.  10, 
could  possibly  be  regarded  as  having  been  repeatedly  infected. 

*  Negative  examinations  were  repeated  in  most  instances. 

t  Moved  into  Bed  2  on  this  day. 


Leo  Buerger 


115 


A  further  series  of  similar  observations  now  follow  in  tabtilated 
form: 

TABLE  X. 

OBSERVATIONS    ON    BED    8. 


Bed  8. 

Positive  Cases. 

Total  No.* 

Dec.    1 8,  neg. 

Beds  I,  2,  4,  9 

4 

"     28,  pes. 

I,  2,  3,  4,  10 

5 

"     29,  pes. 

I,  2,  3,  4,  10 

S 

"     30.  neg. 

I,  2,  3,  4,  10 

5 

Jan.      4,  pes. 

I.  2,  3,  4,  5,  10 

6 

"      10,  neg. 

i,t  2,  3,  4,  5,  6 

6 

"     15,  pes. 

— 

"     18,  pos. 

5 

I 

*  Of  other  positive  cases  in  the  ward. 

t  A  new  case  in  the  same  bed,  also  positive. 

The  first  column  shows  the  occasional  absence  and  appearance 
of  pneumococci  in  the  mouth  of  the  patient  in  Bed  8.  In  the 
second  column  have  been  indicated  the  "positive"  cases  in  the 
ward  on  the  corresponding  dates. 

A  similar  series  of  notes  was  made  in  regard  to  Bed  5  of  the 
same  ward.  The  patient  was  a  "normal"  case.  Three  of 
the  cases  in  adjoining  beds  had  pneumococci  in  their  mouths. 
The  case  in  question  probably  acquired  them  in  the  ward. 

TABLE  XI. 

OBSERVATION    ON    BED    5. 


Beds. 

Positive  Cases. 

Total  No. 

Dec.    27,  neg. 

Beds 

I,  2,  4,  10 

4 

Jan.      4,  pos. 

I,  2,  4,  8,  10 

5 

8,  neg. 

* 

9,  pos. 

— 

"     10,  pos. 

I,  2,  3,  4,  6 

S 

"     II,  neg. 

— 

"     IS.  neg. 

8— 

"      18,  pos. 

~ 

*  Blank  means  that  there  was  no  record  of  the  other  ward  cases  on  this  day 
or  on  the  days  immediately  preceding  or  following. 

In  addition  similar  observations  on  two  other  patients  may  be 
cited.     The  patient  in  Bed  6  was  repeatedly  negative  for  almost 


116  Studies  of  the  Pneumococcus  and  Allied  Organisms 

a  week  after  admission  to  the  ward.  At  that  time  patients  in 
Beds  I,  2,  4,  and  lo  had  been  regularly  positive,  the  two  last 
being  cases  of  pneumonia.  Cases  in  Beds  3,  5,  and  8  were  oc- 
casionally positive.  On  January  9th  pneumococci  were  also 
found  in  case  Bed  6.     Two  days  later  they  had  disappeared. 

The  second  case  was  in  Bed  3.  He  was  admitted  about  the 
same  time  as  the  above  case  and  was  free  from  pneumococci. 
To  his  left  were  Beds  i  and  2,  occupied  by  regularly  positive 
cases,  and  to  his  right  a  convalescing  pneumonia  patient.  Nine 
days  after  admission  the  organism  was  detected  in  the  mouth  of 
this  patient. 

The  next  study  was  carried  out  on  a  "normal"  patient  who 
was  regularly  positive,  and  consisted  in  the  examination  of  the 
following  parts  and  articles :  Culture  from  his  dry  lips,  handker- 
chief No.  I,  handkerchief  No.  2,  sputum  cup,  culture  from  the 
lip  of  his  drinking-cup. 

The  usual  mouth  cultures  had  given  positive  results  on  Decem- 
ber 27,  28,  January  i,  6,  7,  8,  and  9th  (Case  36,  Tables  II  and 
III).     E  15  was  isolated  and  studied. 

On  January  7th  a  culture  from  the  dry  lips  showed  typical 
pneumococci.  The  patient's  handkerchief,  after  twenty-four 
hours'  use  (and  when  perfectly  dry),  was  examined  for  the 
presence  of  pneumococci.  Numerous  colonies  of  this  organism 
were  obtained.  Culture  E  20  was  isolated.  The  handkerchief 
was  kept  in  a  sterile  jar  and  examined  thirty-six  hours,  and 
seven  days  later,  and  pneumococci  were  cultivated  on  these 
dates.  On  January  loth  a  second  handkerchief  was  examined 
with  positive  results.  A  sputum  cup  which  was  filled  with  5  % 
carbolic-acid  solution,  on  the  surface  of  which  some  of  the 
patient's  saliva  was  floating,  was  examined  on  January  7th. 
Cultures  from  the  saliva  showed  typical  pneumococci.  A  suc- 
cessful attempt  was  made  to  discover  the  organism  on  the 
patient's  dishes.  Cultures  taken  from  the  lip  of  a  drinking-cup 
some  five  minutes  after  he  had  partaken  of  cocoa  gave  pneimio- 
cocci  (E  22). 

Of  the  observations  made  on  the  larger  male  ward  two  in- 
stances only  will  be  given,  relating  to  two  patients  who  had  been 


Leo  Buerger  .      117 

negative  for  some  time  before  the  pneiimococci  finally  appeared. 
In  the  case  of  the  patient  in  Bed  19,  fifteen  days  elapsed  before 
the  organism  appeared;  in  the  case  of  the  one  in  Bed  20,  a  full 
month.  6  Both  of  these  patients  in  all  probability  acquired  the 
pneumococcus  from  other  cases  in  the  same  ward. 


TABLE 

XII. 

OBSERVATION 

ON 

BEDS    19 

AND    20. 

Bed.  19. 

Bed  20. 

Jan.   II,  neg. 

"     15.     " 
"     16,      " 

Dec.  27,  neg. 
Jan.   II, 
"     16,      " 

"     19.     " 
"     26,  pos. 
"     27,     " 
"     29,  neg. 

"     27,  pos. 

The  conclusions  in  regard  to  communicability  which  I  have 
reached  are : 

(i)  "Normal"  individuals  in  whose  mouths  the  pneumococcus 
is  repeatedly  found  to  be  absent  may  acquire  the  organism  by 
association  with  cases  of  pneumonia  or  with  "positive  normal" 
persons;  (2)  the  handkerchiefs  and  dishes  of  pneumonia  and 
"positive  normal"  cases  may  be  regarded  as  means  of  trans- 
portation of  the  pneumococcus  from  person  to  person. 

STUDY    OF    THE    PNEUMOCOCCI    ISOLATED. 

The  characters  of  the  pneumococcus  having  already  received 
so  much  attention  at  the  hands  of  previous  workers,  much  of 
the  following  study  will  necessarily  be  repetition.  A  point  of 
especial  importance  was  the  relationship  of  the  pneumococcus  in 
the  normal  mouth  to  the  organism  obtaiaed  from  pneumonia 
cases  and  from  other  pathological  processes.  In  the  study  of  the 
pneumococcus  in  the  normal  mouth  and  in  the  mouths  of  pneu- 
monia cases,  I  have  considered  the  typical  characteristics  and  the 
variations  which  occur  among  a  large  number  of  different  strains 
of  pneumococci.    I  observed  the  morphology,  cultural  properties, 

'  It  is  of  course  recognized  that  positive  results  might  have  been  obtained 
earlier  by  taking  cultures  at  shorter  intervals. 


118  Studies  of  the  Pneumococcus  and  Allied  Organisms 

metabolic  activity,  pathogenicity,  and  agglutinative  action  of 
the  organisms,  studied  comparatively  the  organisms  from  the 
two  sources,  and  the  best  means  of  isolating  the  pneumococci, 
and  considered  what  should  be  the  determining  factors  in  their 
identification. 

Table  II  shows  the  sources  of  the  organisms  studied  in  this 
place,  but  in  addition  ten  other  strains,  nine  of  which  were 
isolated  from  cases  of  empyema  and  one  from  a  suppurating 
joint,  were  used  by  way  of  control  in  the  study  of  morphology 
and  cultural  properties. 

Morphology  of  the  pneumococci. — The  changes  in  form,  which  very  many 
strains  undergo  on  the  various  media,  in  exudates  and  blood  of  animals,  and  on 
the  same  medium  from  generation  to  generation  may  be  very  great.  It  is 
therefore  of  importance  in  a  comparative  study  to  observe  the  organisms  under 
conditions  which  shall  be  as  nearly  alike  in  each  instance  as  possible.  In  most 
cases  the  growths  on  Loeffler  serum,  and  the  colonies  on  the  plates  were  ex- 
amined with  the  capsule  method  from  eighteen  to  twenty-four  hours  after  the 
cultures  were  taken.  The  first  generation  of  each  pneumococcus  strain  could, 
as  a  rule,  be  regarded  as  having  developed  a  morphology  more  or  less  char- 
acteristic for  it. 

The  types  of  pneumococci  regularly  met  with,  excluding  involution  forms,  may 
be  grouped  in  the  following  way:  the  typical  form,  the  small  form,  the  large 
form,  the  bacillary  form,  and  the  streptococcus  form. 

The  typical  form  presents  lancet  or  less  commonly  coccoid-shaped  elements 
enclosed  in  a  characteristic  capsule.  The  capsule  takes  the  form  of  a  deeply 
staining  peripheral  ring,  elliptical  in  contour,  and  separated  from  the  body  of 
the  organism  by  a  considerable  interval.  If  we  designate  this  outer  ring  as  the 
"capsular  membrane,"  the  substance  included  between  it  and  the  cocci  may  be 
termed  "capsular  substance."  The  latter  may  remain  perfectly  clear  and  un- 
stained, or  take  on  a  faint  color.  When  these  forms  are  met  with  in  culture, 
degenerated  and  empty  capsules  as  well  as  typical  chains  are  frequently  present. 

The  degenerated  capsules  stain  poorly,  or  may  present  tears  in  their  periphe- 
ral membrane.  The  empty  forms  also  stain  faintly.  Some  of  them  appar- 
ently contain  diplococci ;  others  have  lost  one  of  the  two  cocci.  The  capsule  at 
the  end  of  a  chain  may  be  empty,  one,  two,  or  more  cocci  being  absent.  When 
the  pneumococci  come  into  contact  there  is  often  a  fusion  of  the  capsules,  with 
a  disappearance  of  the  "capsular  membrane"  where  they  join.  The  typical 
pneumococcus  chain  is  composed  of  capsule  and  elements  similar  to  those  de- 
scribed for  the  diplococcus.  Usually  a  slight  constriction  between  the  diplo- 
cocci is  to  be  seen.  The  cultures  of  pneumococci  vary  considerably  as  to  the 
presence  of  the  degenerate  and  empty  capsules  and  chain  forms.  At  times 
typical  encapsulated  diplococci  preponderate;  at  other  times  many  of  the 
other  varieties  are  present  in  large  numbers. 

The  small  form  possesses  a  capsule  which  is  narrow,  with  a  more  delicate 


Leo  Buerger  119 

contour,  and  with  a  capsular  membrane  approaching  the  body  of  the  organism 
more  closely.  Many  strains  show  this  type  in  the  very  first  generations.  The 
larger  pneumococci  just  described  may  degenerate  into  smaller  forms  after  a 
number  of  generations,  even  on  the  best  culture  media.  When  transplanted  on 
dry  or  unfavorable  media  they  often  present  this  picture. 

The  larger  forms  are  characterized  more  by  the  size  and  development  of  their 
capsules  than  by  any  marked  increase  in  the  size  of  the  enclosed  cocci.  The 
capsules  are  broader,  not  so  well  limited,  and  more  diffusely  staining  than  the 
usual  type.  Further,  they  have  a  tendency  to  break  up  in  the  process  of  pre- 
paring the  specimen,  due  to  their  greater  viscosity.  This  type  of  organism  may 
resemble  the  diplococcus  forms  of  the  Streptococcus  mucosus. 

In  a  number  of  instances  I  have  found  strains  whose  members  are  elongated 
so  as  to  form  veritable  bacilli.  Many  of  these  are  due  to  fusion  of  smaller  ele- 
ments. However,  this  mode  of  origin  of  the  bacillary  forms  is  often  not  ap- 
parent. The  capsules  are  generally  narrow  and  diffusely  stained.  The  type 
belongs  to  the  luxuriantly  growing  pneumococci.  It  may  appear  in  the  first  gen- 
erations directly  isolated  from  the  mouth,  or  in  the  later  generations  of  the 
"large,"  more  mucoid  type.  Short  bacillary  forms  are  very  common.  The 
typical  lancet-shaped  cocci,  if  slightly  elongated,  are  of  this  variety. 

The  streptococcus  type  is  characterized  by  elements  which  are  round  or 
irregular  in  shape.  Capsules  may  be  absent.  When  they  are  present  they 
tend  to  form  delicate  membranes  situated  very  close  to  the  cocci  and  to  sur- 
round the  single  members  rather  than  the  pairs.  I  have  elsewhere  referred  to 
this  type  of  capsule  as  the  "streptococcus  type."  '  At  times  even  the  first 
generations  show  this  arrangement.  These  encapsulated  chains  are  frequently 
found  as  degenerations  of  pneumococci  which  have  been  transplanted  a  great 
number  of  times  or  have  been  cultivated  on  unfavorable  media. 

The  following  table  includes  the  "completely  studied"  pneumococci.  The 
characteristic  morphology  in  the  first  generation  and  especial  points  in  mor- 
phology which  manifested  themselves  in  later  cultures  are  indicated. 

TABLE  XIII. 

MORPHOLOGY  OF  THE  PNEUMOCOCCI.* 

E     I,  typical  f  8,  typical. 

2,  "        ;  or  elongated.  9,  " 

3,  "  i°>  " 

4,  "  II,  typical;  at  times  elongated,  and 

5,  typical  on  Loeflfler;    early  cul-  bacillary. 

tures  on  serum-agar  were  of  12,  typical. 

the      bacillary     type  ;      four  13,  " 

months  after  isolation  typical  14,  " 

forms  were  found.  15,  typical;  or  elongated. 

6,  typical.  16,  " 

7,  small.  17,  typical. 

*  For  the  sake  of  reference  a  number  of  streptococci  are  included  in  this  list, 
t  "Typical,"  "small,"  etc.,  refer  to  the  types  of  organisms.    Whenever  nothing 
to  the  contrary  is  stated  the  encapsulated  form  is  to  be  understood. 

'  Proceedings  of  the  New  York  Pathological  Society,  1904,  iv,  131. 


120  Studies  of  the  Pneumococcus  and  Allied  Organisms 


TABLE  XIII  (Continued). 


i8, 

19. 

20, 
21, 
22, 
23. 
24. 
25. 
26, 

27. 

28, 
29, 

(30. 

(31, 
32, 
33- 
34, 
35, 
36, 
37, 
38- 
39, 
40, 


typical;  or  elongated. 

(41, 
(42, 

typical. 

typical;  or  elongated. 

typical. 

43: 

44 
45 

small;    streptococcus  form  with 

its  typical  capsule, 
typical. 

46: 

Streptococcus  mucosus.)** 
atj^pical  form.)** 
typical. 

47: 

48: 

49: 
5O: 

typical;  large  capsules, 
large,  mucoid-type. 

typical. 

mostly  non-encapsulated;  some 

chains  with  streptococcus  type 

of  capsule. 

**  Considered  in  the  section 


SI, 

52, 
53, 
54, 

55, 

56, 

(57. 


Streptococcus  mucosus.) 

atypical  organism.)** 

small;  or  streptococcus  type 
with  the  streptococcus  type  of 
capsule. 

typical. 

small;  or  streptococcus  type 
with  the  streptococcus  type  of 
capsule. 

atypical;  but  with  large  cap- 
sules; involution  forms  with 
well  developed  capsules. 

typical. 


small. 

typical  and  elongated. 

typical. 

many    pneumococcus    type    of 

chains;   and  typical  forms, 
typical. 


streptococcus . )  *  * 


on  "allied"  organisms. 


From  the  above  table  it  will  be  seen  that  most  of  the  pneumo- 
cocci  showed  the  typical  encapsulated  or  slightly  elongated 
forms.  A  few  showed  the  small  and  bacillary  type,  and  two 
the  large  mucoid  forms.  The  streptococcus  variety  was  foimd 
in  three  cultures.  Although  the  particular  morphology  above 
indicated  in  the  table  was  noted  in  the  first  generation  of  each 
culture,  either  on  the  Loeffier  senun  or  the  senun-glucose-agar 
plates,  it  must  be  mentioned  that  many  strains  either  lost  their 
capsules  entirely  after  a  number  of  transplantations  or  became 
converted  into  other  types.  It  appeared  that  most  of  them 
could  assume  the  smaller  or  streptococcus  type  under  certain 
conditions  of  growth.  The  bacillary  and  large  forms  were  at 
times  replaced  by  the  typical  forms.  None  of  the  varieties, 
however,  changed  their  morphology  to  the  large  mucoid  type. 
Degenerative  and  involution  changes  occurred  in  some  cultures 
of  all  the  strains. 

The  cultural  properties. — All  the  completely  studied  organisms  were  inocu- 
lated on  the  following  culture  media:  broth  neutral  to  phenolphthalein,  neutral 
agar,  Loeffler's  blood-serum,  gelatin,  litmus  milk,  and  Hiss'  inulin-serum  water. 

Broth:  All  the  strains  with  the  exception  of   E  8  caused  visible  growth  in 


Leo  Buerger 


121 


the  bouillon.  The  luxuriance  and  the  rapidity  of  growth  varied  greatly. 
Some  strains  (E  5)  developed  a  heavy  and  diffuse  turbidity  in  twenty-four 
hours ;  others  produced  only  the  faintest  cloud.  Very  few  pneumococci  caused 
the  formation  of  flakes  in  either  turbid  or  clear  bouillon. 

Agar:  Most  of  the  organisms  grew  poorly  on  this  medium.  E  37  and  38 
gave  very  profuse  growths.  They  presented  appearances  characteristic  of 
Streptococcus  mucosus. 

Loefiier's  blood-serum:  The  usual  type  of  growth  characteristic  for  pneumo- 
cocci was  generally  obtained.  There  was  considerable  variation  as  to  the 
luxuriance  and  watery  character  of  the  surface  growths.  E  5,  37,  and  38  de- 
veloped rapidly ;  the  first  gave  watery,  and  the  other  two  mucoid,  colonies. 
Some  cultures,  however,  were  rather  attenuated  and  their  colonies  fairly  dry. 

Gelatin:  At  24°  C.  most  of  the  strains  finally  gave  evidences  of  growth.  At 
best  the  colonies  were  very  fine  and  the  development  extremely  sparse.  There 
was  never  any  liquefaction.  The  following  organisms  gave  no  perceptible 
growth:  E  11,  12,  13,  20,  29,  45,  53,  and  54.  All  of  these,  with  the  exception 
of  E  45  and  54,  were  virulent  for  white  mice.  The  statement  made  by  some 
observers,  to  the  effect  that  the  a  virulent  pneumococci  do  not  develop  on 
gelatin  (at  the  ordinary  temperatures)  whereas  the  virulent  forms  do,  could 
not  be  confirmed. 

Litmus  milk:  The  time  required  for  acid  production  to  manifest  itself  and 
for  coagulation  to  take  place  were  noted  in  each  instance.  The  results  are  given 
in  the  following  table. 

TABLE  XIV. 


GROWTH  IN 

LITMUS  MILK 

E. 

ac* 

coag.t 

E. 

ac* 

coag.t 

E. 

ac* 

coag  t 

I 

I 

3 

19 

I 

2 

39 

I 

2 

2 

4 

20 

" 

'  * 

40 

** 

3 

*  * 

" 

21 

* ' 

" 

42$ 

3 

4 

" 

" 

22 

^ 

I^** 

43 

s 

2 

23 

2 

3 

44 

2 

6 

2 

10? 

24 

I 

4 

45 

" 

7 

I 

4 

25 

" 

46 

3 

8 

— 

26 

2 

47 

2 

9 

** 

2 

27 

" 

48 

4 

10 

2 

3 

28 

" 

49 

I 

II 

I 

2 

29 

" 

50 

2 

12 

" 

*' 

32 

" 

51 

3 

13 

3 

33 

I 

52 

** 

14 

2 

34 

2 

53 

" 

IS 

" 

i§ 

35 

" 

54 

— 

16 

" 

I 

36 

** 

55 

' 

2 

17 

2 

37 

" 

18 

38 

3 

*  ac.  =  acid  in  as  many  days  as  indicated.  f  coag.  =  coagulated. 

**  About.  X  Atypical  organism;   see  section  on  allied  organisms. 

Two  or  three  days  was  the  usual  time  required  for  coagulation.  Three  strains, 
E  8,  43,  and  54,  produced  a  moderate  amount  of  acid  but  no  coagulation. 

Inulin-serum  water :  Table  No.  XV  shows  in  how  many  days  coagulation 
set  in. 


122 


Studies  of  the  Pneuraococcv^  and  Allied  Organisms 


TABLE  XV. 

GROWTH    IN    INULIN-SERUM  WATER. 


E. 

coag. 

E. 

coag. 

1 

E. 

coag. 

E. 

coag. 

I 

4 

i 

;       ^5 

2 

1 

1         29 

2 

45 

2 

2 

'^ 

i        i6 

I         1 

!     31* 

5 

46 

*' 

3 

** 

17 

2            i 

1     32 

2 

47 

" 

4       . 

*' 

i8 

4 

2>Z 

I 

48 

* ' 

5      ' 

" 

19 

2 

34 

2       1 

49 

3 

6 

2 

!            20 

" 

35 

'  * 

50 

2 

7 

'* 

;       21 

*' 

36 

'* 

i        51 

3 

8 

" 

22 

I 

37 

3 

52 

2 

9 

5 

1        23 

4 

38 

" 

53 

*^ 

10 

2 

i            24 

I 

39 

2 

54 

5 

II 

I 

25 

3 

!        40 

3        ' 

55 

2 

12 

3 

,            26 

" 

1        42* 

5 

13 

2 

27 

2 

43 

3 

14 

28 

5 

44 

2 

*  Atypical  organism. 

Coagiilation  as  a  rule  took  place  in  from  two  to  fotir  days.  Acid  production 
could  often  be  noted  in  one  or  two  days.  Although  this  medium  was  in  main 
sufficiently  favorable  for  the  growth  of  pneiimococci,  a  number  of  lots  of  serum 
water  were  made  up  which  had  to  be  discarded.  These  last  were  found  to 
furnish  a  poor  medium  for  the  development  of  the  organism.  It  is  difficult  to 
determine  the  reasons  for  the  variations  in  the  quality  of  the  medium.  An 
attempt  was  made  to  use  beef-serum  which  was  free  from  red  blood  cells  and 
in  which  very  Httle,  if  any,  haemolysis  had  taken  place.  The  various  lots  were 
tested  by  inociilation  with  luxuriantly  growing  pneiimococci.  If  acid  produc- 
tion and  coagulation  were  deferred  for  a  number  of  days  the  medium  was  re- 
placed by  a  fresh  lot. 

I  foiuid  that  the  serum-water  medium  may  be  made  more  favorable  for  the 
growth  of  pneumococci  by  the  addition  of  peptone.  A  modified  serum-water 
medium  was  prepared  as  follows:  Hiss'  serum-water  mixttire  is  first  made  up. 
One  hundred  c.c.  of  beef-serum  and  200  c.c.  of  distilled  water  are  put  into  a 
flask  and  steamed  for  ten  minutes.  A  peptone  solution  is  prepared  by  dissolving 
6  grams  of  Witte's  peptone  in  25  to  30  c.c.  of  water  over  a  small  flame.  When 
dissolved  it  is  filtered  and  cooled.  The  peptone  mixtxire  is  then  added  to  the 
serum  water.  To  this  medium  as  a  basis  i  %  of  intilin  and  sufficient  litmus 
are  added  in  the  usual  way.  Kahlbaum's  litmus  has  given  me  the  most  satis- 
factory results.  Both  the  peptone  solution  and  the  serum  water  should  be 
cold  before  mixing.  The  medium  is  sterilized  for  15  minutes  on  three  or  four 
successive  days.  After  sterilization  it  has  a  thin  jelly-like  consistency.  Acid 
production  by  pneumococci  is  usually  manifested  in  18  to  24  hotirs. 

All  of  the  pneumococci  studied  fermented  inulin.s     The  action  of  certain 

8  A  number  of  strains  failed  to  ferment  inulin  in  certain  generations.  Others 
caused  only  a  suspicion  of  acid  in  the  inulin-serum  water,  but  gave  a  more 
marked  reaction  medium  as  modified  bv  me. 


Leo  Buerger  123 

organisms  resembling  the  pneumococcus  on  inulin  will  be  considered  in  a 
separate  section. 

Colonies :  I  wish  to  call  attention  to  a  number  of  points  regarding  the  colo- 
nies of  the  pneumococcus.  I  have  already  referred  to  the  characteristic  "ring" 
appearance  that  many  strains  present.  As  seen  in  the  serum-glucose-agar 
plates,  the  "ring-forms"  vary  both  in  size  and  structure.  Thus  a  central  um- 
bilication  is  sometimes  to  be  discerned  on  colonies  as  small  as  the  period  em- 
ployed in  ordinary  newspaper  type.  On  the  other  hand,  I  have  occasionally 
seen  the  colonies  attain  a  diameter  of  1.5  to  almost  2  mm.  The  colonies  of  this 
type,  when  dense,  lack  the  characteristic  milky  appearance  given  to  them  by 
transmitted  light.  They  are  then  more  mucoid  and  whiter  in  appearance. 
The  central  umbilication  may  not  become  manifest  until  48  hours  have  elapsed, 
so  that  a  slightly  convex  watery  or  mucoid  colony  may  show  the  typical  appear- 
ance if  it  be  incubated  or  allowed  to  remain  at  room  temperatiire  24  hours 
longer. 

There  are  both  small  and  large  colonies  which  do  not  show  the  ring  form. 
The  small  colonies  are  slightly  convex,  glistening,  and  of  varying  opacity. 
They  may  be  watery  or  even  somewhat  mucoid.  Very  large  colonies  are  occa- 
sionally observed.  These  may  be  mistaken  for  the  colonies  of  the  Streptococcus 
mucosus  capsulatus.  Their  development  is  just  as  great  and  general  character- 
istics the  same.  The  colonies  of  E  37  and  E  38  attained  a  diameter  of  2.5  mm. 
on  the  surface  of  the  plates.  Both  strains  were  isolated  from  a  case  of  pneu- 
monia. As  far  as  the  macroscopic  appearance  of  these  cultures  was  con- 
cerned they  were  indistinguishable  from  the  mucoid  streptococcus.  E  33  also 
gave  colonies  almost  as  large  as  those  just  described.  After  a  number  of  trans- 
plantations all  three  strains  showed  a  marked  diminution  in  the  luxvuiance  of 
their  growth  on  serum  media. 

PATHOGENICITY    OF    THE    PNEUMOCOCCI. 

The  question  of  the  virulence  of  the  organisms  isolated  from 
the  mouths  of  the  "completely  studied"  cases  was  determined 
by  inoculating  white  mice  with  approximately  uniform  doses 
of  the  pure  cultures.  The  rapid  diminution  in  virulence  which 
the  pneumococcus  suffers  after  a  number  of  transplantations, 
the  difficulty  in  employing  exact  dosage,  and  the  slight  variations 
in  the  weight  of  the  mice  employed,  are  facts  which  affect  the 
interpretation  of  the  results.  It  was  possible,  in  most  instances, 
to  inject  pure  cultures  but  one  generation  removed  from  the 
original  isolation.  Of  fifty-one  (51)  pneumococci  derived  from 
the  mouth,  there  were  five  in  which  later  generations  were  used. 
The  five  strains,  however,  were  virulent  even  after  the  second 
generation.  These  studies,  therefore,  may  be  regarded  as  a  fair 
index  of  the  pathogenicity  of  the  organisms. 


124 


Studies  of  the  Pneumococcus  and  Allied  Organisms 


The  usual  dose  was  a  twenty-four-hour  growth  on  a  serum-agar 
slant.  This  was  suspended  in  i  c.c.  of  normal  salt  solution  and 
inoculated  subcutaneously  in  the  back  of  a  white  mouse.  When- 
ever the  results  were  negative  or  doubtful,  as,  for  example,  the 
possible  death  of  the  mouse  from  some  other  cause,  fresh  ani- 
mals were  employed,  or  larger  and  repeated  doses  were  some- 
times injected  into  the  same  animal.  In  some  instances  the 
attempt  was  made  to  convert  avirulent  into  virulent  strains. 

The  following  table  deals  with  the  virulence  of  fifty-one  pneu- 
mococci  isolated  from  the  mouth : 


TABLE  XVI. 

VIRULENCE    OF    THE    PNEUMOCOCCI. 


Strain. 

Period  of  Survival. 

Generation 

Strain. 

Period  of  Survival. 

Generation 

E     I 

I  day. 

2d 

E27 

I   day  16  hrs. 

2d 

2 

I     "      21  hrs. 

" 

28 

I      "      12    " 

3 

3  days 

3d 

29 

I      "      IS    " 

4 

2      "        2  hrs. 

2d 

32 

—     —  22    " 

S 

I  day    12    " 

" 

U 

—     —15    " 

6 

avirulent. 

" 

34 

—     —  22    " 

7 

" 

" 

35 

2  days  12    " 

8 

I  day    18  hrs. 

" 

36 

I  day    14    " 

9 

"      "       "    " 

' ' 

37 

—     — 22    " 

lO 

avirulent. 

" 

38 

3  days  12    " 

3d 

II 

I  day    16  hrs. 

" 

39 

—     — 18    " 

12 

"      "      18   " 

" 

40* 

(—     —19?" 

8th) 

13 

"      "      21    " 

" 

43 

avirulent. 

2d 

14 

20    " 

44 

—    — 23    " 

IS 

2  days  12    " 

45 

avirulent. 

16 

"     "      16    " 

" 

46 

I  day    18  hrs. 

" 

17 

3      "      12    " 

" 

47 

avirulent. 

18 

avirulent. 

" 

48 

I  day    14  hrs. 

19 

4  days  12  hrs. 

" 

49 

avirulent. 

20 

I  day    24    " 

" 

50 

2  days  15  hrs. 

21 

2  days  19    " 

" 

51 

I  day    16    " 

22 

avirulent. 

" 

52 

—     — 22    " 

23 

9  days  16  hrs. 

" 

53 

I  day      2    " 

24 

avirulent. 

" 

54 

avirulent. 

25 

I  day    18  hrs. 

3d 

55 

2  days. 

26 

3  days  12    " 

2d 

*  Doubtful  result;   additional  mice  were  negative. 

The  virulent  strains  varied  considerably  in  the  rapidity  with  which  they 
brought  about  the  death  of  the  animal.  The  shortest  time  was  is  hours. 
Most  of  the  mice,  however,  succumbed  within  two  or  three  days.  The  patho- 
logical changes  consisted  of  fibrinous  exudate  with  varying  amounts  of  hjemor- 
rhage  and  oedema  at  the  site   of  inoculation,   and  now  and  then  increased 


Leo  Buerger  125 

peritoneal  or  pleuritic  fluid.  The  typical  encapsulated  diplococci  were  found 
regularly  in  films  made  from  the  exudate  and  the  blood.  In  a  few  instances 
the  diplococcus  was  also  present  in  spreads  raade  from  the  peritoneal  or  pleural 
fluid.  At  times,  especially  in  the  case  of  strains  E  37  and  38,  the  exudate  was 
of  a  mucoid  character,  but  never  as  gelatinous  as  in  the  case  of  the  Strepto- 
coccus mucosus  capsulatus.  When  death  did  not  set  in  rapidly  (23)  more 
marked  lesions  were  observed,  such  as  peritonitis,  fibrinous  perihepatitis, 
pleuritis,  and  slight  splenic  enlargement.  Congested  or  hasmorrhagic  areas  in 
the  lungs  were  but  rarely  seen. 

Cultures  were  taken  from  the  blood  and  local  exudate  of  every  mouse  which 
died  of  the  infection,  and  the  organisms  again  identified  by  the  capsule  stain. 

The  avinilent  pneumococci,  although  injected  in  much  larger 
doses,  produced  no  appreciable  symptoms  in  the  test  animals. 
In  each  of  these  cases  at  least  two  mice  were  employed.  In  a 
number  of  the  eleven  negative  instances  intraperitoneal  or  sub- 
cutaneous injections  into  mice  were  employed,  and  the  animals 
killed  in  eighteen  to  twenty -four  hours.  Capsule  stains  were 
then  made.  Thus  E  40  was  avirulent  for  most  of  the  animals 
and  did  not  present  the  typical  morphology  on  the  plates, 
but  it  showed  non-encapsulated  and  degenerate  forms  of  the 
pneumococcus  as  represented  by  chains  with  or  without  the 
"streptococcus  type"  of  capsule.  An  inoculated  mouse  killed 
twenty-four  hours  after  injection  showed  typical  but  small  en- 
capsulated pneumococci. 

Of  the  total  of  fifty -one  cultures  of  mouth  pneumococci,  eleven 
were  avirulent  and  forty  virulent,  or  21.5  %  and  78.5  %,  re- 
spectively. In  the  cases  of  the  "normal"  mouth  cultures,  eight 
were  avirulent  out  of  thirty-eight,  or  21  %.  Of  thirteen  cultures 
from  the  pneumonia  cases,  three  were  avirulent,  or  23  %.  Ap- 
parently, therefore,  about  the  same  proportion  of  non-virulent 
pneumococci  are  to  be  found  in  the  mouths  of  patients  suffering 
from  pneumonia  as  in  the  mouths  of  normal  individuals. 

Pneumococci  occtirring  in  the  mouths  of  normal  individuals 
are,  as  far  as  we  have  been  able  to  determine,  identical  with 
those  occurring  in  the  mouths  of  patients  with  pneumonia  and 
obtained  from  other  sources.  A  few  minor  differences  were 
noted.  Pneumococci  isolated  from  the  mouth  grow  more 
rapidly  and  more  luxuriantly  than  those  isolated  from  pus  of 
emp3^£ema.     The   elongated   almost   bacillary  types  with  well- 


126  Studies  of  the  Pneuraococcvis  and  Allied.   Organisms 

formed  capsules  occurred  more  frequently  in  the  recent  cultures 
from  "normal"  cases.  This  latter  peculiarity  appears  to  be  an 
expression  at  times  of  high  development  under  favorable  cul- 
tural conditions.  In  regard  to  virulence  and  the  lesions  pro- 
duced in  white  mice  no  differences  were  to  be  noted.  Some 
strains  from  the  "normal"  mouth  retained  their  pathogenicity 
quite  as  long  as  the  strains  from  other  sources.  Thus  culture 
E  5,  from  a  "  normal"  case,  was  still  virulent  almost  four  months 
after  isolation. ^ 

SUMMARY    ON    THE    OCCURRENCE    OF    PNEUMOCOCCUS. 

1 .  Certain  characteristic  morphological  types  of  pneumococcus 
were  met  with  regularly  in  the  course  of  this  study. 

2.  The  pneumococci  occurring  in  the  mouths  of  normal  in- 
dividuals possess  the  usual  morphological  and  cultural  characters 

observed  in  the  organisms  isolated  from  other  sources. 

3.  All  the  pneumococci  studied  possessed  the  power  of  splitting 
inulin  with  the  formation  of  acid. 

4.  The  percentage  of  virulent  organisms  found  in  the  mouths 
of  normal  cases  was  79  ;  in  pneumonia  cases  77. 

ORGANISMS     IN    THE    MOUTHS     OF    NORMAL     INDIVIDUALS     WHICH 
CLOSELY    RESEMBLE    THE    PNEUMOCOCCUS. 

In  this  section  will  be  considered  certain  organisms  which  be- 
long to  the  pneumococci  but  present  variations  from  the  usual 
type,  organisms  related  to  the  pneumococci  but  possibly  inter- 
mediate in  development  between  the  streptococci  and  pneumo- 
cocci, and  the  Streptococcus  mucosus  capsulatus. 

Certain  atypical  pneumococci: — Culture  E  40  presented  none  of  the  typical 
capsulated  forms    of   the    pneumococcus    at  the  time  of    isolation,  but  non- 

9  In  ten  cases  the  saliva  of  the  patient  was  taken  for  direct  animal  inocula- 
tion at  the  same  time  that  plates  were  made.  The  intention  was  to  compare 
the  value  of  the  plate  method  and  animal  inoculation  in  recovering  the  pneumo- 
coccus. By  the  latter  means  four  positive  instances  were  obtained.  In  each 
of  these  the  pneumococcus  was  isolated  in  pure  culture,  ready  for  the  test  in- 
oculation, at  the  end  of  forty-eight  hoiors.  Only  three  positive  cases  were 
secured  by  direct  animal  injection.  It  was,  moreover,  necessary  to  plate  out 
the  organism  from  the  blood  of  the  infected  animals  in  order  to  obtain  pure 
cultures.  It  would  seem,  therefore,  that  more  reliable  and  rapid  results  are 
to  be  obtained  not  by  inoculation  but  by  the  plate  method. 


Leo  Buerger  127 

capsulated  diplococci  and  chains  with  the  streptococcus  type  of  capsule. 
Several  mice  inoculated  with  early  generations  of  the  organism  were  not  affected 
One  mouse  died  nineteen  hours  after  inoculation  with  the  eighth  generation,  but 
as  no  organisms  were  found  in  the  blood  this  result  was  regarded  as  doubtful. 
Typical,  although  poorly  developed,  capsular  forms  were,  however,  discovered 
in  a  mouse  killed  for  the  purpose  of  diagnosis. 

This  organism  was  found  variable  in  its  action  on  inulin.  A  number  of  tests 
were  made  with  different  generations  and  with  a  strain  isolated  from  a  mouse. 
After  three  days  there  was  slight  coagulation  with  moderate  amount  of  acid 
production.  Complete  coagulation  did  not  take  place.  At  times  no  apprecia- 
ble acid  was  formed.  One  tube  showed  the  reaction  at  the  end  of  six  days. 
The  distinguishing  features  of  this  organism  were:  atypical  morphology,  non- 
\'irulence,  and  variable  fermentative  power  for  inulin. 

Of  the  pneumococci  -ndth  large  mucoid  colonies  three  cultures  were  isolated 
from  the  mouth.  A  similar  strain  was  cultivated  from  the  exudate  in  a  case  of 
early  empysema.  These  pneumococci  are  to  be  distingtiished  from  the  Strepto- 
coccus mucosus  by  their  morphology.  Their  elements  are  regularly  lancet- 
shaped  in  the  early  generations.  Their  chains  are  of  the  pneumococcus  type, 
i.  e.,  composed  of  pairs  of  lancet-shaped  cocci  surrounded  by  a  capsule  which 
shows  a  tendency  to  slight  constriction  between  the  diplococci.  The  Strepto- 
coccus mucosus  under  the  same  conditions  will  be  found  to  possess  either  round 
or  biscuit-shaped  elements,  and  the  caps\ile  about  its  chains  never  shows  the 
indentation  so  common  in  the  case  of  pnetmiococci.  The  luxuriance  of  growth 
of  these  mucoid  pneumococci  may  diminish  rapidly  after  a  number  of  trans- 
plantations. The  Streptococcus  mucosus,  on  the  other  hand,  does  not  seem  to 
undergo  much  change. 

Organisms  related  to  the  pneumococcus: — Culture  E  31  was  isolated  from  the 
mouth  of  a  so-called  normal  patient.  Its  morphology  was  peculiar  and  quite 
different  from  that  of  the  ordinary  pneumococcus.  Specimens  stained  with 
the  capstde  method,  and  taken  from  cultures  on  serum-agar,  or  Loeffler's  medium, 
presented  the  following  picture.  Most  of  the  elements  were  grouped  in  pairs, 
and  were  coccoid  or  slightly  elongated.  Often  single  cocci  or  short  bacillary 
forms  with  rounded  ends  were  to  be  seen.  The  capsules  when  they  enclosed 
two  elements  often  showed  deep  constrictions  between  the  cocci,  so  that  the 
latter  appeared  almost  separated.  Another  feature  was  the  tendency  for  the 
organisms  to  clump.  When  aggregated  in  this  way,  the  contiguous  capsules 
fused  at  the  lines  of  contact,  so  that  a  single  enveloping  membrane  for  a  whole 
clump  could  be  seen.  Even  considerable  manipulation  in  the  process  of  spread- 
ing failed  to  separate  them  completely.  1°  In  neutral  or  serum -bouillon  long 
chains  were  formed. 

The  following  was  noted  in  cultures:  Milk  was  coagulated  in  five  days. 
With  inulin  it  behaved  variably.  The  early  cultures  caused  fermentation,  the 
later  ones  fermented  slowly  or  not  at  all.  Mouse  No.  i  received  four  inocula- 
tions without  effect.  Mouse  No.  2  died  in  forty-three  hours;  there  was  a 
fibrinous  hemorrhagic  exudate  locally.  No  typical  pneumococcus  forms  were 
found  either  in  the  exudate  or  in  the  blood.     Mouse  No.  3  did  not  succumb. 

10  This  appearance  became  somewhat  altered  after  long  cultivation. 


128  StMdies  of  the  Pneumococcus  and  Allied  Organisms 

Mouse  No.  4  was  killed  for  diagnosis,  but  no  organisms  were  detected  in  the 
spreads.  This  strain  was  therefore  of  doubtful  virulence.  The  features  of  the 
organism  were :  peculiar,  atypical  morphology;  irregular  action  on  inulin,  and 
non-virulence  for  mice. 

Still  another  type  (E  42)  resembling  the  pneumococcus  was  isolated  from  a 
"normal"  patient.  On  Loeffler-serum  and  serum-agar  media  it  grew  as  diplo- 
cocci  which  showed  very  delicate  eccentrically  placed  capsules.  Usually  only 
one  half  of  the  capsule  was  discernible.  Careful  adjustment  of  the  focus,  and 
the  use  of  a  high  ociolar,  often  enabled  one  to  see  the  other  side  of  the  capsule 
as  a  faint  linear  envelope  situated  very  close  to  the  bodies  of  the  diplococci. 
A  similar  appearance  was  now  and  then  to  be  seen  in  the  case  of  short  chains. 

Cultural  characters:  Milk  was  coagulated  in  four  days,  neutral  bouillon  was 
diffusely  clouded;  on  gelatin  there  was  very  poor  growth.  Variable  results 
were  obtained  on  the  inulin  medium.  Two  of  the  early  cultures  coagulated  the 
inulin-serum  water  in  four  and  five  days  respectively.  At  other  times  only  a 
very  little  acid,  or  none  at  all,  was  produced.  Animal  inoculations  were  nega- 
tive. The  typical  pneumococcus  form  was  not  formed  even  after  a  number  of 
animal  inoculations.  This  organism  may  therefore  be  regarded  as  having 
features  in  common  both  with  the  streptococcus  and  the  pneumococcus. 

Streptococcus  mucosus  capsulatus: — A  number  of  organisms  belonging  to  this 
group  were  isolated  from  normal  mouths.  They  form  a  distinct  class  by  them- 
selves and  should  be  differentiated  from  pneumococci,  although  the  latter,  both 
in  their  cultiu-al  properties  and  morphology  may  often  closely  resemble  them. 

In  the  blood  and  exudates  of  infected  animals  their  characteristic  mor- 
phology is  developed.  In  these  the  Streptococcus  mucosus  takes  the  form  of 
diplococci  or  chains  siirrounded  by  a  particular  type  of  capsule.  The  elements 
are  usually  somewhat  biscuit-shaped.  However,  they  may  be  coccoid,  slightly 
elongated,  or  even  lancet-shaped.  The  lancet  form  is  rare,  and  when  it  is  found 
is  usually  associated  with  many  of  the  other  forms.  The  capsules  of  the  diplo- 
cocci are  almost  circular  in  outline,  very  large  and  broad,  with  a  tendency  to 
stain  diffusely.  Those  surrounding  the  chains  give  no  evidence  of  having  been 
formed  by  the  fusion  of  smaller  elements.  The  outer  membrane  shows  none 
of  the  indentations  which  are  commonly  seen  on  the  pneumococcus  chains.  In 
the  blood  and  exudates  of  infected  animals  the  diplococcus  form  usually  pre- 
ponderates. Short  chains  of  four  are  very  common  and  chains  of  six  or  eight 
elements  may  be  found. 

On  culture  media,  especially  on  the  serum  media  and  Loeffier's  coagulated 
blood-serum,  the  same  morphology  is  to  be  observed.  Although  the  capsules 
of  the  streptococcus  are  fairly  resistant  in  the  early  generations  taken  from 
animal  exudates,  in  general  they  are  more  easily  broken  up,  more  mucoid,  and 
less  well  defined  in  their  outlines  than  those  of  the  typical  pneumococcus.  In- 
volution forms,  longer  chains,  and  smaller  and  more  membranous  envelopes 
usually  occur  in  the  later  generations. 

The  colonies  of  these  organisms  on  the  plates  often  attain  considerable  dimen- 
sions. As  a  rule,  they  measure  about  1.5  to  2  mm.,  are  mucoid  and  glassy  by 
reflected  light,  milky  and  more  opaque  by  transmitted  light.  Colonies  raeasur- 
ing  3  mm.  or  slightly  more  in  diameter  are  occasionally  seen.  If  the  plates  are 
allowed  to  remain  at  room  temperature  for  a  second  24  hours  after  incubation. 


Leo  Buerger  129 

an  appearance  similar  to  that  presented  by  the  "ring  type"  of  pneumococcus 
colonies  is  often  developed.  The  centers  of  the  colonies  become  clear  and  the 
peripheries  stand  out  as  milky  annular  bands. 

On  the  surface  of  the  serum-agar  or  Loeffler's  medium  there  is  a  glistening, 
mucoid,  apparently  transparent  growth.  Discrete  colonies  may  be  likened  to 
drops  of  water.     By  transmitted  light  the  opacity  of  the  colonies  can  be  seen. 

Milk  and  the  inulin-serum  water  are  coagulated  rapidly.  The  growth  on 
gelatin  is  poor,  and  there  is  no  liquefaction. 

The  organisms  studied  were  virulent  for  white  mice,  death  usually  resulting 
in  20  to  48  hours.  After  subcutaneous  inoculation  a  mucoid,  almost  gelatinous 
exudate,  often  with  considerable  oedema  and  hcemorrhage,  was  produced  locally. 
The  typical  encapsulated  organisms  were  present  in  the  exudate  and  in  the 
blood. 

Although  the  very  profuse  and  characteristic  growth  of  this  organism  on 
serum-agar  or  serum-glucose -agar  usually  enables  one  to  differentiate  it  from 
the  pneumococcus,  this  is  not  always  the  case.  Reference  has  already  been 
made  to  three  strains  of  pneumococci  as  they  grew  on  the  above  mentioned 
media  which  were  indistinguishable  from  the  Streptococcus  mucosus.  For 
the  diagnosis  one  must  rely  upon  morphological  differences.  The  forms  in 
the  blood  and  exudate  of  animals  inoculated  with  the  two  types  of  organisms 
show  the  same  differences  in  morphology  as  do  their  cultures  on  favorable 
media.  After  a  number  of  transplantations,  the  pneumococci  may  suddenly 
lose  their  luxuriance  of  growth,  while  the  mucoid  streptococci  vary  but  little  in 
this  respect.  In  older  cultures  the  distinction  of  the  organisms  by  means  of 
their  morphology  presents  no  difficulties. 

RESUME    OF   THE  FACTS    RELATING  TO    IDENTIFICATION  AND  CHIEF 
DIAGNOSTIC    FEATURES    OF   THE    PNEUMOCOCCUS. 

The  identification  and  isolation  of  the  pneumococci  were  accomplished  by  the 
plate  method  and  on  Loeffler's  serum.  The  organisms  which  could  be  identified 
as  pneumococci  by  means  of  their  morphology  were  (a)  typical  encapsulated 
forms,  (&)  small  encapsulated  forms,  (c)  large  encapsulated  forms,  (d)  bacillary 
encapsulated  forms;  other  organisms  encountered  were  non-encapsulated 
diplococci  or  chains  ";  encapsulated  streptococci,  i.  e.,  streptococci  with  the 
streptococcus  type  of  capsule;  diplococci  or  chains  with  atypical  capsules,  or 
Tsrith  other  peculiar  morphological  characters;  streptococci  with  mucoid  cap- 
sules (Streptococcus  mucosus  capsulatus). 

Pneumococci  classified  as  "typical,"  "small,"  large,"  or  "bacillary,"  when 
virulent,  regularly  presented  the  typical  morphology  in  the  blood  and  exudates 
of  white  mice.  Positive  animal  tests  were  regarded  as  furnishing  conclusive 
evidence  as  to  the  nature  of  the  organism.  From  a  study  of  a  large  number  of 
strains  it  was  found  that  organisms  of  the  above  morphology  always  belonged 
to  the  pneumococci.  Even  when  not  virulent  it  was  possible  to  demonstrate 
the  typical  encapsulated  forms  in  animals. 

11  Any  of  these  typical  varieties  (a),  {b),  (c),  and  {d)  may  grow  in  chains,  and 
possess  the  characteristic  capsule.  The  word  "chains"  as  used  here  does  not 
refer  to  these  types. 


130  Studies  of  the  Pneumococcus  and  Allied  Organisms 

The  value  of  the  morphological  identification  on  Loeffler's  serum  was  inves- 
tigated by  plating  out  about  twenty-five  cultvxres  and  controlling  the  results  in 
that  way.  Piire  cultures  of  pneumococci  were  obtained  in  all  of  the  tubes  in 
which  encapsulated  pneumococci  had  been  recognized  in  the  films. 

In  a  separate  study  it  was  found  that  pneumococci  frequently  lost  their  cap- 
sules after  a  number  of  transplantations  or  when  cultivated  on  tmfavorable 
culttire  media.  Non-encapsulated  chains  resembling  streptococci  were  also 
met  with  under  these  conditions.  The  question  arose,  therefore,  as  to  whether 
the  organism  could  possibly  lose  its  typical  form  in  the  first  generation  on 
Loeffler's  blood-serum  or  on  serum-glucose-agar  plates,  and  take  on  the  form 
of  non-encapsulated  diplococci  and  chains.  A  large  number  of  these  forms 
were  therefore  isolated  and  studied  both  culturally  and  by  animal  inoculation. 
None  of  these  could  be  converted  into  the  encapsulated  types  or  identified  with 
the  pneumococcus  by  cultural  or  other  tests.  It  would  seem,  therefore,  that 
pneumococci  isolated  from  the  mouth  and  growing  on  the  media  employed, 
regularly  take  on  the  encapsulated  form  in  the  first  generation. 

Encapsulated  streptococci  with  spherical  or  biscuit-shaped  elements  and 
surrounded  by  a  membranous,  linear  envelope  12  were  more  frequently  met 
with  especially  on  the  Loeffler  serum.  These  occur  frequently  as  degeneration 
forms  of  pneumococci,  but  may  belong  to  the  streptococci.  A  number  of  such 
streptococci  were  isolated  and  studied,  E  26  and  45  showed  this  character, 
but  their  identification  was  easy  on  account  of  the  presence  of  the  "small" 
encapsulated  pneumococcus  forms.  E  40,  however,  showed  none  of  the  varie- 
ties of  diplococci  which  are  at  once  recognizable  by  their  morphology.  There 
were  many  encapsulated  chains  of  the  "streptococcus  type,"  but  the  typical 
diplococcus  form  was  obtained  in  a  white  mouse.  Of  the  many  other  encap- 
sulated streptococci  isolated  and  studied  none  could  be  proven  to  be  pneumo- 
cocci, either  culturally  or  by  animal  inoculation.  There  was,  therefore,  only 
one  instance  (E  40)  in  which  the  pneumococcus  did  not  show  some  one  or  other 
of  the  typical  pneumococcus  forms  in  the  first  cultures.  Possibly  poor  quality 
of  the  medium  might  explain  this  case.  1  ^ 

There  seems  little  doubt  that  there  are  many  doubtful  diplococci  of  atypical 
morphology  possessing  characters  in  the  stained  specimens  which  are  somewhat 
different  from  those  of  the  ordinary  pneumococci,  and  yet  in  cultures  they  may 
exhibit  characters  resembling  both  the  pneumococci  and  the  streptococci. 
E  31  and  42,  which  have  already  been  described  at  length,  differed  from  the 
pneumococci  in  their  morphology  and  resembled  them  in  their  ability  to  fer- 
ment inulin.  Animal  inoculations  in  these  cases  failed  to  give  any  positive  evi- 
dence of  their  pneumococcal  nature.  One  strain  resembling  E  31  and  another 
resembling  E  42  had  previously  been  isolated  but  not  completely  studied.  At 
the  present  time  I  am  unable  to  classify  these  two  strains.  Perhaps  they  are 
intermediate  organisms  between  streptococci  and  pneumococci.  Possibly  E  42 
approaches  nearer  the  streptococci  and  E  3 1  the  pneumococci. 

The  Streptococcus  mucosus  belongs  to  a  group  which  is  related  to,  but  dis- 
tinct from,  the  pneumococcus.  Its  recognition  by  means  of  morphology  is 
usually  easy. 

1'  I.  e.,  the  "streptococcus  type"  of  capsule. 

»3  E  57  was  a  streptococcus  which  showed  a  narrow  capsule. 


Leo  Buerger  131 

As  one  becomes  acquainted  with  the  various  types  of  pneumococci  when 
stained  with  the  capsule  method,  their  differentiation  from  other  encapsulated 
organisms,  such  as  the  Micrococcus  tetragenus,  the  diplococcoid  forms  of  the 
Friedlander  bacillus,  and  the  encapsulated  diplococcus  often  found  in  beef- 
serum,  presents  little  difficulty.  The  capsules  of  Micrococcus  tetragenus  are 
usually  small, '^  and  the  cocci  themselves  large  and  round  or  flattened.  The 
Friedlander  bacillus  is  identified  by  the  employment  of  the  combined  ' '  Gram 
and  capsule"  stain.  The  organism  found  in  beef -serum  is  a  somewhat  elon- 
gated stout  coccus  which  is  much  larger  than  the  pneumococcus,  and  has  a 
narrow  membranous  capsule. 

Perhaps  the  most  valuable  cultural  features  in  the  diagnosis  of  the  pneumo- 
coccus are  the  fermentation  of  inulin,  the  growth  on  serum-glucose-agar,  and 
the  appearances  of  the  colonies. 

In  a  study  of  the  streptococci  it  was  found  that  a  certain  number  of  these 
are  able  to  split  inulin  with  the  formation  of  acid.  The  present  work  confirms 
the  view  of  Hiss  that  all  pneumococci,  as  far  as  known,  ferment  this  carbo- 
hydrate. Some  pneumococci,  however,  fail  to  produce  acid  or  coagulate  the 
inulin-serum  water  in  certain  generations.  This  variation  has  been  frequently 
noted,  and  to  a  certain  extent  impairs  the  value  of  the  test. is  Continued 
failure  to  ferment  inulin  would  seem  to  speak  against  an  organism  being  the 
pneumococcus,  while  the  possession  of  the  fermenting  power  cannot  be  regarded 
as  proving  it  to  be. 

Finally,  as  regards  the  serum-glucose-agar  medium,  it  may  be  added  that 
practically  all  streptococci  caused  precipitation  or  whitening  of  it.  The  pneu- 
mococci, on  the  other  hand,  with  very  few  exceptions,  showed  no  tendency  to 
produce  this  change. »« 

AGGLUTINATION    OF    THE    PNEUMOCOCCI    AND    ALLIED    ORGANISMS. 

In  this  section  I  will  present  my  obsen^ations  on  the  agglu- 
tination of  many  of  the  pneiimococci  described  in  the  previous 
sections.  Unfortunately,  time  did  not  permit  an  exhaustive 
study  of  this  phase  of  the  subject.  Although  a  sufficiently  large 
number  of  strains  were  studied,  only  a  very  small  number  of 
sera  could  be  obtained.    The  study  embraced  the  following  topics : 

Agglutination  of  pneumococci  by  immune  senmi. 

Agglutination  of  allied  organisms  and  streptococci  by  the 
serum  of  animals  immunized  against  the  pnetmiococcus. 

»*  I  have  recently  isolated  a  strain  of  the  Micrococcus  tetragenus,  whose  cap- 
sules are  of  the  large  mucoid  type.  This  organism  is,  however,  easily  recog- 
nized. 

>  s  The  variations  in  the  quality  of  the  medium  and  the  growth  of  the  culture 
must  be  taken  into  consideration.  A  single  negative  experiment  is  of  little 
value;   it  should  be  confirmed  by  repeated  tests. 

J  6  Libman,  op.  cit.,  has  made  similar  observations. 


132  Studies  of  the  Pneumococcus  and  Allied  Organisms 

Agglutination  of  pneiunococci  by  means  of  the  serum  of 
patients  with  pneumonia. 

Agglutination  of  pneumococci  from  so-called  normal  mouths 
by  the  serum  of  the  persons  from  whom  they  were  isolated. 

Agglutination  of  the  pneumococci  with  immune  serum,. — For  the  purpose  of 
immunization  pneumococci  from  two  sources  were  selected:  strains  isolated 
from  pus  from  an  empyema,  and  from  the  "normal"  mouth.  Rabbits  were 
inoculated  intravenously  or  intravenously  and  subcutaneously  together.  The 
cultures  injected  consisted  of  serum-agar  growths  incubated  for  twenty-four 
hours,  and  suspended  in  normal  saline. 

Six  rabbits  were  immunized  with  as  many  different  strains.  Two  of  these 
succumbed  after  several  weeks.  Rabbit  No.  i  was  inoculated  dtiring  a  period 
of  four  months  and  showed  marked  emaciation.  Its  serum  never  developed 
any  marked  agglutinating  power.  The  same  was  true  of  all  the  animals,  with 
the  exception  of  rabbit  No.  3.  The  serum  of  this  animal  was  found  to  have 
'  attained  a  higher  value  than  any  of  the  rest,  and  was  selected  for  most  of  the 
reactions.  Culture  E  5  was  employed  for  the  immunization  of  this  animal. 
After  a  period  of  about  two  months  and  a  half  this  rabbit  also  succximbed. 
Sufficient  serum  had  been  obtained  to  carry  on  a  number  of  experiments. 

In  the  preparation  of  cultures  for  agglutination  it  was  decided  to  employ 
bacterial  suspensions  in  normal  (0.85  %)  saline  rather  than  broth  or  serum- 
broth  ciilttires.  After  considerable  experimentation  the  following  procedtore, 
similar  to  one  employed  by  Wadsworth.i?  was  adopted. 

Small  flasks  (or  a  number  of  large  tubes)  of  serum-bouillon  is  (neutral  bouillon 
3  parts,  ascitic  serum  i  part)  were  inoculated  and  incubated  for  twenty-four 
hoturs.  The  bacterial  cells  were  then  centrifuged,  washed  twice  by  means  of 
normal  saline,  and  the  sediment  thoroughly  shaken  up  in  some  of  this  fluid. 
Inasmuch  as  there  was  considerable  variation  in  the  luxuriance  of  growth  of 
the  various  strains,  it  was  not  possible  to  adopt  any  hard  and  fast  rules  as  to 
the  quantity  of  the  salt  solution  to  be  used  for  the  emulsion.  The  proper 
density  of  the  suspensions  having  been  determined  by  a  series  of  tests,  fresh 
tubes  of  suspensions  were  kept  on  hand  as  controls  or  standards. 

Serum-bouillon  was  selected  in  preference  to  the  plain  broth  because  it  was 
found  to  be  much  more  favorable  for  the  growth  of  the  organisms.  Although 
the  early  generations,  and  recent  isolations  from  animals,  regularly  gave  fairly 
luxuriant  cultures  in  plain  broth,  the  later  cultures  often  developed  poorly. 
Most  of  the  strains  had  been  kept  alive  on  artificial  media  for  a  considerable 
time  before  agglutination  tests  were  made.  In  the  serum-broth  no  albuminous 
precipitation  could  be  detected  at  the  end  of  twenty-four  hours.  Microscopic 
examination  of  the  saline  suspensions  showed  practically  nothing  but  the 
bacterial  cells.  It  was  found,  however,  that  the  serum-bouillon  was  not  always 
suitable  for  this  work.      Certain  of  the  pneumococci  did  not  produce  a  diffuse 

''T Jour,  of  Medical  Research,  1903-04,  x,  228. 

1 8  The  broth  was  made  from  meat  infusion  which  had  been  neutralized  before 
boiling,  and  contained  2  %  peptone. 


Leo  Buerger  133 

turbidity  in  this  medium,  but  grew  with  the  formation  of  dense  fliocculi.''  The 
flakes  being  very  difficult  to  break  up,  the  process  of  washing,  sedimenting,  and 
suspending  failed  to  separate  the  clumps  completely.  Suspensions  made  from 
these  cultures  had  therefore  to  be  discarded. 

Pneumococci  which  grew  in  fiocculi  were  cultivated  on  a  number  of  serum- 
agar  slants,  from  which  the  surface  growth  was  suspended  in  saline  and  treated 
in  the  same  way  as  the  bouillon  cultures.  A  satisfactory  distribution  of  the 
organisms  was  obtained  in  this  way.  A  number  of  tests  sufficed  to  show  that 
the  agglutinability  of  the  agar-  and  serum-bouillon  cultures  was  practically  the 
same. 

The  macroscopic  reactions  were  made  in  the  usual  way.  One  half  c.c.  of  a 
sufficiently  dense  suspension  was  added  to  a  similar  volume  of  the  dilute  serum. 
Dilutions  were  made  up  to  750  or  1000,  the  tubes  incubated  at  37°  C,  and  ob- 
served for  twenty-four  hours.  Generally  the  macroscopic  reactions  are  not 
absolutely  trustworthy.  The  clumping  very  often  does  not  manifest  itself  in 
the  higher  dilutions  until  eight  to  twelve  hours  have  elapsed.  At  about  this 
time  a  certain  amount  of  sedimentation  by  gravity  may  occur  in  the  controls. 
At  the  end  of  twenty-four  hours  this  has  become  even  more  marked,  so  that  it 
is  often  impossible  to  decide  as  to  the  value  to  be  put  on  the  test.  Moreover, 
the  clumps  may  be  so  fine  as  to  escape  observation.  This  may  interfere  with 
the  decision  both  as  to  time  and  to  the  dilution  in  which  the  reaction  sets  in. 
The  microscopic  reactions  are  not  open  to  these  objections. 

The  microscopic  reactions  were  made  in  every  instance,  very  often  side  by 
side  with  macroscopic  tests.  At  least  two  sets  of  reactions  were  made  with 
each  organism.  In  one  the  dilutions  were  freshly  made,  and  in  the  other  stock 
dilutions  of  the  serum  were  employed.  The  results  were  practically  the  same 
in  both  instances.  A  small  drop  of  the  culture  was  mixed  with  a  small  drop  of 
the  dilute  serum  on  a  cover-slip  for  examination  by  the  hanging-drop  method. 
These  slides  were  incubated  just  as  were  the  tubes.  Microscopic  examinations 
of  the  contents  of  the  tubes  were  also  made.  The  results  of  these  often  differed 
considerably  from  the  macroscopic  readings. 

On  account  of  the  limited  amounts  of  serum  and  the  large  number  of  pneumo- 
cocci to  be  tested,  I  was  unable  to  carry  out  the  macroscopic  tests  on  all  the 
strains.  A  sufficient  number  were,  however,  made  to  assure  me  that  the 
microscopic  tests  were  in  main  just  as  reliable  as  the  macroscopic  ones. 

The  reactions  with  the  senim  of  Rabbit  3  alone  will  be  given. 
A  month  after  the  first  intravenous  inociilation,  the  serum  of 
this  animal  agglutinated  neither  streptococci  nor  pneumococci. 
Gradually  increasing  agglutinative  power  was  developed.  Just 
before  the  death  of  the  animal,  the  serum  had  attained  its  maxi- 
mum power.  The  rabbit  was  bled  while  moribund,  the  blood 
collected,  and  the  serum  stored  in  the  refrigerator.  At  that 
time  it  was  tested  with  the   homologous  pneumococcus   E  5. 

19  The  same  is  true  of  the  plain  neutral  broth. 


134  Studies  of  the  Pneumococcus  and  Allied  Organisms 


M 

i 

+++++++ 
+  +  +  +  + 
+  +  +  +  + 
1 

ji 

+ 

+  +  +  +   1     1     1     1     1 

+ 

;J;+  I  1  1  1  1  1  1 

A 

+++++ 1  1  1  1 

"^ 

++ 1  1  1  1  1  1  1 

H 

+++++++ 
+++++++ 1  1 
+++++++ 

d 

+++++++ 
+++++++ 1  1 
+++++++ 

^ 
-* 

+++ 
++++++ 

O, 

i 

++++ 

+++    1  1  1  1  1 

++ 

X. 

++ 

^^+  1  1  1  1  1  1 

1  1 

•i 

+ 

++ 1 1 1 1 1  1  1 

+ 

Ok 

+  +  +  +  +  +  T; 

^ 

,J3 

++++ 
1  1  i 

o  o  o  o  o  o 

100*00100    o    0>0 
w   lot^M    M    (N    colore 

TS 


:3 

a 

o 

o 

<D 

o 

^ 

+-> 

+-> 

o 

O 

PI 

W) 

> 

Pi 

^ 

03 

^ 

<U 

4J 

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PI 

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a 

Leo  Buerger 


135 


Complete  agglutination  was  obtained  in  a  dilution  of  1:150. 
The  study  of  the  greater  number  of  strains  was  deferred  for  a 
month,  at  which  time  the  value  for  E  5  had  dropped  to  i :  100. 

Additional  macroscopic  reactions  were  made  with  pneumo- 
cocci  E  6  to  E  12  inclusive.  The  results  were  practically  the 
same  as  those  obtained  in  the  microscopic  tests.  The  following 
reactions  were  obtained  in  from  twelve  to  twenty -four  hours. 


^Complete. 

Incomplete. 

Complete. 

Incomplete. 

E    6 

7 
8 

9 

1:75 
1 :  100 
1:50 
1:7s 

1 :  200 

i:  150 
1 :  100 

E  10 
II 
12 

1:50 
1:50 
1:50 

1 :  100 
1:75 

By  the  microscopic  tests  agglutination  can  often  be  observed  in  twenty  or 
thirty  minutes.  The  temperature  of  37°  C.  seems  to  hasten  the  process.  Par- 
tial or  even  complete  reactions  may  take  place  in  much  higher  dilutions  at  37° 
C.  than  at  room  temperature. 

The  appearance  of  true  clumping  under  the  microscope  is  very  characteristic. 
When  the  agglutination  is  complete,  the  diplococci  or  chains  are  approximated 
in  irregular  areas  which  are  bound  together  by  strands  or  isthmuses  of  clumped 
bacteria.  The  clumps  are  not  discrete  as  in  the  case  of  the  typhoid  bacillus. 
They  have  a  tendency  to  join  by  means  of  narrow  offshoots  or  processes.  As 
a  rule,  such  large  masses  include  practically  all  the  bacteria.  More  rarely 
we  find  a  number  of  free  non-agglutinated  organisms  and  small  discrete  clumps. 
When  agglutination  is  incomplete,  larger  or  smaller  masses  of  pneumococci  are 
found  scattered  throughout  the  field.  These  features  were  also  found  to  occur 
with  the  streptococci. 

In  table  No.  XVIII  the  results  of  the  agglutination  reactions 
of  pneumococci  from  the  following  sources  are  given :  (a)  normal 
mouths;  (6)  the  mouths  of  pneumonia  patients;  (c)  empyaema 
pus.  The  readings  show  the  reactions  at  the  end  of  twelve  to 
sixteen  hours.  Observations  were  regularly  continued  until 
twenty -four  hours  had  elapsed.  Usually  there  was  very  little 
difference,  if  any,  between  the  sixteen-hour  and  twenty-four- 
hour  reactions. 

The  sera  of  three  normal  rabbits  were  tested  against  fifteen 
pneumococci  and  all  the  streptococci  and  atypical  organisms. 
The  dilutions  were  made  from  i :  5  up  to  i :  750.  In  no  instance 
was  there  evidence  of  any  agglutinative  reaction  in  these  dilutions. 


136 


Studies  of  the  Pneumococcus  and  Allied  Organisms 


TABLE  XVIII. 

MICROSCOPIC    AGGLUTINATION    OF    PNEUMOCOCCI    WITH    IMMUNE    SERUM. 


Culture. 

I ;  20 

1 :  40. 

i:  60. 

I ;  100. 

i:  ISO- 

1 :  200. 

1:300. 

i:Soo. 

1:750. 

P   I 

+  +  + 

+  +  + 

+  +  + 

+  +  + 

-  +  +  + 

-  +  +  + 

-  +  +  + 

+ 

P  15 

+  +  + 

+  +  + 

+  +  + 

+  + 

+  + 

— 

— 

— 

— 

PS30* 

+  +  + 

+  +  + 

+  + 

+  + 

+  + 

— 

— 

— 

— 

P  60 

+  +  + 

+  +  + 

+  +  + 

-  +  +  + 

+ 

— 

— 

— 

— 

P  61 

+  +  + 

+  +  + 

+  +  + 

+  +  + 

+  + 

+ 

— 

— 

— 

P  72 

+  ^  + 

+  +  + 

-  +  +  + 

•+  + 

+  + 

+ 

— 

— 

— 

E  I 

+  +  + 

+  +  + 

+  + 

+  + 

+  + 

— 

— 

— 

— 

3 

+  +  + 

-  +  +  + 

-  +  +  + 

-  +  +  + 

-  +  +  + 

-  +  +  + 

+  + 

— 

— 

4 

-  +  +  + 

-  +  +  + 

-  +  +  + 

+ 

— 

— 

— 

— 

— 

5 

+  +  + 

+  +  + 

+  +  + 

+  +  + 

+  + 

+  + 

+ 

+ 

— 

6 

+  +  + 

+  + 

+ 

+ 

— 

— 

— 

— 

— 

7 

+  +  + 

+  +  + 

+  +  + 

+  +  + 

+  +  + 

— 

— 

— 

— 

8 

+  +  + 

+  +  + 

+  + 

+  + 

+  + 

+ 

— 

— 

— 

10 

+  +  + 

+  +  + 

+  + 

+ 

— 

— 

— 

— 

— 

11 

+  +  + 

+  +  + 

— 

— 

— 

— 

— 

— 

— 

12 

+  +  + 

+  +  + 

+  +  + 

+ 

— 

— 

— 

— 

— 

15 

+  +  + 

+  +  + 

+  +  + 

+  + 

— 

— 

— 

— 

— 

16 

+  +  + 

+  +  + 

+  + 

— 

— 

— 

— 

— 

— 

17 

+  +  + 

+  +  + 

+  + 

— 

— 

— 

— 

— 

— 

18 

+  +  + 

-  +  +  + 

+  + 

+ 

— 

— 

— 

— 

— 

19 

+  +  + 

+  +  + 

+  + 

— 

— 

— 

— 

— 

— 

20 

+  +  + 

-  +  +  + 

— 

— 

— 

— 

— 

— 

— 

21 

+  +  + 

+  +  + 

+  +  + 

+  + 

— 

— 

— 

— 

— 

22 

+  +  + 

+  +  + 

+  +  + 

+  +  + 

+  + 

+ 

— 

— 

— 

23 

+  +  + 

+  +  + 

+  + 

+ 

— 

— 

— 

— 

— 

24 

+  +  + 

+  +  + 

+  +  + 

-  +  +  + 

+  + 

+ 

— 

— 

— 

26 

+  +  + 

-  +  +  + 

+  + 

+ 

— 

— 

— 

— 

— 

27 

+  +  + 

+  +  + 

+ 

— 

— 

— 

— 

— 

— 

28 

+  +  + 

+  +  + 

-  +  +  + 

+ 

— 

_ 

— 

— 

— 

29 

+  +  + 

-  +  +  + 

+  + 

— 

— 

— 

— 

— 

— 

32 

+  +  + 

-  +  +  + 

+ 

— 

— 

— 

— 

— 

33 

+  +  + 

-  +  +  + 

+ 

— 

— 

— 

— 

— 

— 

34 

+  +  + 

+  +  + 

+  + 

+ 

— 

— 

— 

— 

— 

35 

-  +  +  + 

-  +  +  + 

+  + 

+ 

+ 

— 

— 

— 

— 

36 

+  +  + 

+  +  + 

+  + 

— 

— 

— 

— 

— 

— 

38 

+  +  + 

+  +  + 

+  +  + 

+  + 

+  + 

— 

— 

— 

— 

39 

+  +  + 

+  +  + 

+  +  + 

+  +  + 

+ 

— 

— 

— 

— 

40 

+  +  + 

+  +  + 

+  +  + 

+  +  + 

-  +  +  + 

+  + 

— 

— 

— 

44 

+  +  + 

+  +  + 

-  +  +  + 

+ 

— 

— 

— 

— 

45 

+  +  + 

+  +  + 

+  +  + 

+  + 

+ 

+ 

+ 

— 

— 

46 

+  +  + 

+  +  + 

-  +  +  + 

+ 

— 

— 

— 

— 

— 

47 

+  +  + 

+  +  + 

+  +  + 

-  +  +  + 

+  + 

+ 

— 

— 

— 

48 

+  +  + 

+  +  + 

+  +  + 

-  +  +  + 

+  + 

+ 

— 

— 

— 

49 

+  +  + 

+  +  + 

+  +  + 

-  +  +  + 

+  + 

+ 

— 

— 

— 

51 

+  +  + 

+  +  + 

+  +  + 

+ 

— 

— 

— 

— 

— 

52 

+  +  + 

+  +  + 

-  +  +  + 

— 

— 

— 

— 

— 

— 

53 

+  +  + 

+  +  + 

+  +  + 

+  + 

+  + 

+ 

— 

— 

— 

54 

+  +  + 

+  +  + 

-  +  +  + 

+ 

— 

— 

— 

— 

— 

55 

+  +  ^ 

+  +  + 

+  +  + 

+  + 

+  + 

+ 

— 

— 

— 

*  An  organism  of  the  large  mucoid  type. 
+  +  +  =  complete;  all  organisms  clumped. 

+  +  +  =  "  with  a  few  scattered  bacteria.         +  +  =  discrete  large  clumps. 

+  =  small  clumps;  many  pneumococci  not  agglutinated.  —  =  negative. 


Leo  Buerger 


137 


If  the  organisms  be  grouped  according  to  their  source,  the 
restdts  may  be  tabulated  in  the  following  manner : 

TABLE  XIX. 

AGGLUTINATION    OF    PNEUMOCOCCI    FROM    THE    NORMAL    MOUTH. 


Complete. 

Incomplete. 

Complete. 

Incomplete. 

E  3 

1 :  200 

1:300 

E  27 

I  :  40 

1 :  60 

4 

1 :  60 

1 :  100 

28 

1 :  60 

1 :  100 

=; 

1 :  100 

1 :  500 

29 

1 :  40 

1 :  60 

6 

1 :  60 

1 :  100 

39 

1 :  100 

i:  150 

II 

1 :  40 

i:  60 

40 

1 :  150 

1 :  200 

12 

1 :  60 

i:  100 

44 

1 :  60 

1 :  100 

15 

1 :  60 

1 :  100 

45 

1 :  60 

1:300 

16 

1 :  40 

1 :  60 

46 

1 :  60 

1 :  100 

17 

1 :  40 

1 :  60 

47 

1 :  100 

1 :  200 

18 

1 :  40 

1 :  100 

48 

1 :  100 

1 :  200 

19 

1 :  40 

1 :  60 

49 

1 :  100 

1 :  200 

20 

1 :  40 

— 

51 

1 :  60 

1 :  100 

21 

1 :  60 

1 :  100 

52 

1 :  60 

— 

22 

1 :  100 

1 :  200 

53 

1 :  60 

1 :  200 

24 

1 :  100 

1 :  200 

54 

1 :  60 

1 :  100 

26 

1 :  40 

1 :  100 

55 

1 :  60 

1 :  200 

(From.  Pneumonia  Cases) 


(From  Empysema  Pus)  * 


E  I 

1 :  40 

i:  150 

P  I 

1:300 

i:  500 

7 

1 :  150 

— 

15 

1 :  60 

i:  150 

8 

1 :  40 

1 :  200 

30 

1 :  40 

i:  150 

10 

1 :  40 

1 :  100 

60* 

1 :  100 

i:  150 

23 

1 :  4c 

1 :  100 

61 

1 :  100 

1 :  200 

32 

1 :  40 

1 :  60 

72 

1 :  60 

1 :  200 

33 

1 :  40 

1 :  60 

34 

1 :  4c 

1 :  100 

35 

1 :  40 

i:  150 

36 

1 :  40 

1 :  60 

38 

1 :  60 

1 :  150 

*  All  from  empyaema  pus,  except  P  60,  which  was  isolated  from  the  blood  of 
a  mouse  which  had  spontaneously  acquired  a  pneumococcus  infection. 

A  consideration  of  Tables  XVIII  and  XIX  appears  to  teach 
that  although  there  are  considerable  variations  in  the  agglu- 
tinability  of  the  various  strains,  these  are  not  sufficiently  marked 
to  enable  one  to  separate  them  into  classes.  The  homologous 
organism  E  5  was  completely  agglutinated  in  a  dilution  of  i :  100 
and  incompletely  up  to  i :  500.  Other  pneumococci,  however, 
gave  just  as  good  or  better  reactions.  Thus  E  3  was  completely 
clumped  in  a   dilution  of  1:200.     Pneumococcus  P  i,  isolated 


138 


Studies  of  the  Pneumococcus  and  Allied  Organisms 


from  empyaema  pus,  gave  the  best  reactions.  Organisms  de- 
rived from  this  source  and  from  the  mouths  of  pneumonia 
patients  were  agglutinated  in  just  as  high  dilutions  as  those  from 
the  mouths  of  "normal"  individuals. 

The  agglutination  of  allied  organisms  and  streptococci  with  the 
serum  of  animals  immunized  against  the  pneumococcus. — The 
same  immune  serum  was  employed  for  agglutination  tests  on 
the  following  organisms:  atypical  intermediate  organisms,  cer- 
tain streptococci,  and  a  number  of  strains  of  the  Streptococcus 
mucosus  capsulatus.  The  microscopic  tests  appeared  to  give 
very  definite  results.  The  picture  of  the  clumping  is  very 
similar  to  the  one  already  described. 

The  tests  on  streptococci  were  made  in  order  to  determine 
whether  the  immune  pneumococcus  serum  possessed  agglutinins 
for  streptococci  as  well  as  for  pneumococci.  The  results  tabu- 
lated below  will  show  that  doubtful  organisms,  the  streptococcus 
and  the  Streptococcus  mucosus  capsulatus,  were  all  agglutinated 
by  the  serum : 

TABLE  XX. 

AGGLUTINATION    OF    VARIOUS    ORGANISMS. 

Streptococci. 


Culture. 

1 :  20. 

1 :  40. 

1:  60. 

1 :  100 

i:  150- 

1 :  200. 

i:  300. 

1:500. 

1:750. 

S      5 

+  +  + 

+  +  + 

+  +  + 

+  + 

+ 

+ 



_ 

_ 

7 

+  +  + 

+  +  + 

+  +  + 

-  +  +  + 

+  + 

+  + 

+  + 

+  + 

+ 

9 

+  +  + 

+  +  + 

+  +  + 

+  + 

+ 

+ 

— 

— 

— 

II 

+  +  + 

+  +  + 

+  +  + 

+ 

+ 

— 

— 

— 

— 

23 

+  + 

+  + 

+ 

— 

— 

— 

— 

— 

— 

31 

+  +  + 

+ 

— 

— 

— 

— 

— 

— 

— 

80 

+  +  + 

+  +  + 

+  +  + 

+  +  + 

+ 

— 

— 

— 

— 

E  57 

+  +  + 

+  +  + 

-  +  +  + 

+ 

+ 

— 

— 

— 

— 

J 

itypical  ' 

Intermedt 

ate"  Or  go 

misms  an 

d  Streptoc 

occus  Mu 

casus  Group. 

Culture. 

1 :  20. 

1 :  40. 

1 :  60. 

1 :  100. 

i:  ISO. 

1 :  200. 

i:  300 

1:500. 

i:7S0. 

E31 

+  +  + 

+  +  + 

+  +  + 

4- 









_ 

^       42 

+  +  + 

+  +  + 

+  +  + 

+  + 

+ 

— 

— 

— 

— 

S.M.I 

+  +  + 

+  +  + 

+ 

+ 

+ 

— 

— 

— 

— 

II 

+  +  + 

+  +  + 

+  +  + 

+  -f 

+ 

— 

— 

— 

— 

IV 

+  +  + 

+  +  + 

+  +  + 

+ 

+ 

+ 

— 

' 

Leo  Buerger 


139 


TABLE  XXI. 

SUMMARY    OF    AGGLUTINATION    TESTS    OF    TABLE    XX. 


Streptococci. 


Atypical  Organisms. 


Complete. 

Incomplete. 

Complete. 

Incomplete. 

S    5 

1 :  60 

1 :  200 

E31 

1 :  60 

i:  100 

7 

1 :  100 

1:750 

42 

1 :  60 

1:150 

9 

1 :  60 

1 :  200 

23 

— 

i:  60 

Streptococcus  Mucosus. 

31 

i:  20 

1 :  40 

S.  M.  I 

1 :40 

i:  150 

77 

i:  60 

i:  150 

11 

1 :  60 

i:  150 

80 

1 :  100 

i:  150 

IV 

i:  60 

i:  150 

E57 

i:  60 

i:  150 

S  s  and  S  9  were  isolated  from  empyeema  pus;  S  7  and  S  23  from  the  blood  of 
cases  of  "malignant"  endocarditis;  S  31  from  an  appendix;  S  77  and  80  from 
the  intestinal  contents  of  normal  individuals,  and  £57  from  the  "normal" 
mouth.  For  E  31  and  42  see  Table  II.  S.  M.  I  was  isolated  from  the  cerebro- 
spinal fluid  of  a  case  of  cerebro-spinal  meningitis,  S.  M.  II  from  a  spontaneously 
infected  mouse,  and  S.  M.  IV  from  a  "normal  mouth." 

It  appears,  therefore,  that  an  immune  pneumococcus  serum  can 
agglutinate  streptococci,  certain  atypical  organisms,  and  the 
Streptococcus  mucosus  as  well  as  pneumococci,  I  was  unable 
to  study  the  specificity  of  the  agglutinins  by  the  absorption 
methods.  The  tests  made  showed  that  the  agglutination  re- 
action was  of  no  aid  in  the  classification  of  the  pneumococci; 
whether  or  not  an  immune  serum  of  higher  value  would  have 
shown  greater  differences  in  its  reaction  towards  pneumococci 
and  streptococci  I  cannot  say. 

A  few  experiments  were  made  in  order  to  determine  the  differ- 
ence of  agglutinability  of  pneumococci  which  had  been  recently 
recovered  from  an  animal  and  the  old  stock  strains.  At  times 
there  was  no  appreciable  difference.  At  others,  the  recently 
isolated  organism  was  clumped  in  somewhat  higher  dilutions. 
Thus  E  5  was  completely  agglutinated  in  a  dilution  of  1:150 
after  passage  through  one  animal,  whereas  the  stock  culture 
gave  a  reaction  of  i :  100. 

The  agglutination  of  pneumococci  by  the  serum  of  pneumonia 
patients. — The  sera  of  three  cases  were  employed  for  the  tests. 
Two  of  them  were  fairly  fresh  and  were  obtained  from  Cases  31 


140 


Studies  of  the  Pneumococcus  and  Allied  Organisms 


and  45  (see  Table  II).  The  third  serum,  kindly  given  me  by 
Dr.  Libman,  had  been  taken  from  a  very  severe  case  of  lobar 
pneumonia  in  April,  1904,  and  was  therefore  about  eleven  months 
old.     It  had  been  kept  in  the  refrigerator  and  was  sterile. 

The  sera  from  Cases  31  and  45  gave  no  agglutination  reaction 
either  with  the  homologous  organism  (from  the  mouth  of  the 
same  case)  or  with  a  number  of  other  pneumococci. 

The  old  serum,  however,  still  showed  considerable  agglutina- 
tive power.  A  niunber  of  pneumococci  were  tested  with  it; 
some  were  clumped  and  others  gave  no  reaction  at  all,  although 
tested  a  number  of  times.  The  results  of  some  of  the  reactions 
are  given  below : 

TABLE  XXII. 

AGGLUTINATION    WITH    PNEUMONIC    SERUM. 


1 :  20. 

1 :  40. 

i:  60. 

1 :  100. 

i:  ISO. 

1 :  200. 

1:300. 

l:Soo. 

1:750. 

p 

I 

+  +  + 

+  +  + 

+  +  + 

+  +  + 

+  +  + 

+  +  + 

+  + 

+ 

+ 

E 

^ 

+ 

+  + 

+  +  + 

+ 

— 

— 

— 

— 

— 

E 
P 

S 
15 
21 

+  + 

+  + 

+  + 

+ 

— 

— 

— 

— 

— 

- 

- 

- 

- 

- 

- 

— 

— 

— 

It  is  interesting  to  note  that  P  i  gave  the  best  reactions  not 
only  with  the  immune  serum  but  also  with  the  old  pneumonia 
serum.  This  particular  organism  had  been  kept  alive  for  many 
months,  but  still  retained  its  encapsulated  diplococcus  form. 
P  15,  although  regularly  presenting  the  typical  form,  was  not  at 
all  affected  by  the  serum  in  a  number  of  tests. 

Serum  from  a  case  of  uraemia  and  from  a  normal  individual 
served  as  controls  in  these  experiments.  Neither  gave  any 
reaction  in  a  dilution  of  1:5. 

The  agglutination  of  pneumococci  by  the  serum  of  normal  indi- 
viduals.— Sera  from  a  number  of  the  so-called  normal  individuals 
were  obtained  l:)y  aspiration  of  large  veins  in  the  region 
of  the  elbow.  Each  of  these  was  tested  against  the  organ- 
ism derived  from  the  mouths  of  the  same  patient.  Dilutions 
from   1:5  up  to  1 :  500  were  made  and  both  macroscopic  and 


Leo  Buerger  141 

microscopic  tests  set  up.     None  of  the  sera  showed  any  agglu- 
tinating power.     The  tests  are  given  below : 

Case  24  vs.  E  16  and  19;      result  negative. 
"     36  "    E  15,  20,  and  22; 
"     39    "    E  18  and  10  *; 
"     32    "    E  II    "      12  *; 
"     41    "    E  17    "      12  *; 

The  agglutinability  of  the  particular  suspension  was  controlled  by  making 
reactions  with  immune  serum. 

*  These  organisms  did  not  belong  to  the  respective  cases,  but  served  as 
controls. 

RESUME    OF   THE    WORK   ON   AGGLUTINATION. 

The  successful  immunization  of  rabbits  against  pneumococci 
is  attended  with  considerable  difficulty  owing  to  the  marked  sus- 
ceptibility of  these  animals  to  a  general  infection. 

The  animals  elaborate  agglutinins  slowly  and  poorly. 

All  the  pneumococci,  irrespective  of  their  source,  were  ag- 
glutinated by  means  of  immune  serum. 

One  sample  of  pneumococcus  immune  serum  was  found  to 
agglutinate  various  pyogenic  streptococci,  the  atypical  organisms, 
and  several  strains  of  the  Streptococcus  mucosus  capsulatus. 

The  serum  of  a  pneumonia  patient  varied  as  to  its  power  to 
agglutinate  different  pneumococci.  Some  strains  were  agglu- 
tinated, others  not. 

The  sera  of  normal  human  individuals  and  of  normal  rabbits 
possess  no  agglutinating  power  for  pneumococci,  the  atypical 
organisms,  certain  streptococci,  and  the  Streptococcus  mucosus 
capsulatus. 

GENERAL    SUMMARY. 

1.  In  the  course  of  experiments  for  obtaining  pneumococci 
from  the  oral  secretions  of  human  individuals,  the  "  plate  method" 
described  in  this  paper  was  found  to  be  the  most  reliable  method 
for  isolating  pneumococci. 

2.  An  alkaline  two-per-cent,  glucose-serum-agar  was  regarded 
as  the  most  favorable  medium  for  the  rapid  development  of 
colonies  of  the  pneumococcus. 


142  Studies  of  the  Pneumococcus  and  Allied  Organisms 

3.  The  morphological  identification  of  the  pneiimococcus  was 
made  by  means  of  a  special  capsule-stain.  The  recognition  of 
the  organism  was  accomplished  with  precision  and  reliability 
when  present  in  the  blood  and  exudate  of  test  animals,  and  upon 
the  various  culture  media  employed. 

4.  Pure  cultures  of  pneumococci  could  by  the  methods  de- 
scribed be  obtained  within  forty-eight  hours  for  inoculation  into 
animals  for  tests  of  virulence. 

5.  Normal  persons  often  harbor  the  pneumococcus  in  their 
mouths.  Of  the  seventy-eight  cases  examined,  thirty-nine  showed 
the  presence  of  the  pneumococcus. 

6.  In  a  second  and  larger  series  of  studies  on  so-called  normal 
cases  by  means  of  cultirres  made  from  the  throat  on  Loeffier's 
medium,  the  organism  was  detected  seventy-one  times  in  204 
examinations,  or  in  34.8%.  In  145  cases  the  Streptococcus 
mucosus  capsulatus  occurred  eight  times  (5.5  %),  and  the 
Friedlander  bacillus  twenty-one  times  (14.5  %). 

7.  Certain  individuals  may  acquire  the  pneumococcus  in  their 
mouths  in  the  hospital  wards,  and  may  continue  to  harbor  it  for 
a  considerable  period  of  time. 

8.  The  pneumococcus  may  persist  for  days  or  weeks  in  the 
mouths  of  patients  who  have  recovered  from  pneumonia. 

9.  From  a  study  of  patients  in  the  hospital  wards,  certain  con- 
clusions in  regard  to  communicability  were  drawn.  "Normal" 
individuals,  in  whose  mouths  the  pneumococcus  is  repeatedly 
found  to  be  absent,  may  acquire  the  organism  by  association 
with  pneumonia  or  "positive  normal"  cases.  The  handker- 
chiefs and  dishes  of  pneumonia  and  "positive  normal"  cases  are 
to  be  regarded  as  some  of  the  means  of  transference  of  the  or- 
ganism from  person  to  person.  The  lips  of  drinking-cups  and 
the  sputum  or  saliva  in  sputum  cups  were  found  to  contain  living 
and  virulent  pneumococci. 

10.  Certain  characteristic  morphological  types  of  the  pneumo- 
coccus were  regularly  met  with  in  this  study.  These  include  the 
following:  (i)  the  typical,  (2)  the  small,  (3)  the  large,  (4)  the 
bacillary,  and  (5)  the  streptococcus  type  of  the  pnetunococcus. 

11.  Pneumococci  in  the  mouths  of  normal  individuals  possess 


Leo  Buerger  143 

the  morphological  and  cultural  properties  which  are  character- 
istic of  the  same  organisms  when  isolated  from  other  sources. 

12.  All  pneimiococci  possess  the  power  of  fermenting  inulin 
with  the  formation  of  acid,  even  if  not  in  all  generations.  But 
this  holds  true  only  when  the  inulin  serum- water  medium  as 
modified  by  the  writer  is  used. 

13.  A  certain  configuration  of  the  colonies,  designated  by  the 
term  "ring-form,"  when  present  is  diagnostic  of  the  pneumo- 
coccus. 

14.  The  percentage  of  virulent  pneumococci  present  in  the 
mouths  of  normal  persons  was  79;  in  cases  of  pneumonia,  77. 

15.  Pneumococci  of  the  large  mucoid  type,  and  giving  large 
mucoid  colonies,  should  be  distinguished  from  the  Streptococcus 
mucosus  capsulatus. 

16.  There  are  doubtful  diplococci  of  atypical  morphology 
which  can  be  grouped  neither  with  the  pneumococci  nor  with 
the  streptococci,  although  they  possess  many  features  in  com- 
mon with  both. 

1 7 .  The  Streptococcus  mucosus  capsulatus  belongs  to  a  group 
which  is  related  to,  but  distinct  from,  the  pneumococcus. 

18.  Morphological  characters  and  animal  tests  are  of  greatest 
value  in  the  identification  of  the  pneumococcus.  By  the  em- 
ployment of  suitable  culture  media  it  is  possible,  in  most  in- 
stances, to  recognize  the  pneumococcus  by  its  morphology  alone. 
The  chief  cultural  tests  are  the  following :  the  manner  of  growth 
upon  the  serum-glucose-agar,  and  the  modified  inulin-serum 
water,  and  the  appearance  of  the  "ring-type"  of  colony. 

19.  All  pneimiococci,  irrespective  of  their  source,  were  agglu- 
tinated by  means  of  pneumococcus  immune  serum. 

20.  An  immime  pneumococcus  serum  was  fotmd  capable  of 
agglutinating  various  pyogenic  streptococci,  certain  atypical 
organisms,  and  several  strains  of  the  Streptococcus  mucosus 
capsulatus. 

2 1 .  The  serum  of  pneumonia  patients  varied  in  its  power  to 
agglutinate  different  pneumococci.  Some  strains  were  agglu- 
tinated, others  not. 

22.  The  sera  of  normal  individuals  and  of  normal  rabbits 


144  Studies  of  the  Pneumococcus  and  Allied  Organisms 

possess  no  agglutinating  power  for  pneumococci,  the  atypical 
organisms,  certain  streptococci,  and  the  Streptococcus  mucosus 
capstdatus. 

I  wish  to  express  my  indebtedness  to  the  attending  staff  of 
Mt.  Sinai  Hospital  for  their  co-operation.  Much  of  the  material 
was  taken  from  the  wards  of  Drs.  Rudisch,  Meyer,  Koplik,  Brill, 
and  Manges. 

Further,  I  wish  to  thank  Drs.  Mandlebaum  and  Libman  for 
many  kindnesses,  and  Dr.  Alfred  Cohn,  and  Dr.  Hertz,  for  their 
assistance. 


A  COMPARATIVE  STUDY  OF  PNEUMOCOCCI  AND  AL- 
LIED ORGANISMS. 

The  Report  from  the  Central  Laboratory  to  the  Medical 
Commission  for  the  Investigation  of  Acute  Respira- 
tory Diseases  of  the  Department  op  Health 
of  the  City  of  New  York. 

By  PHILIP  HANSON  HISS,  Jr.,  M.D., 
Adjunct  Professor  of  Bacteriology, 

ASSISTED    BY 

John    Harvey   Borden,   M.D.,    and  Clinton    Beecham    Knapp,   M.D., 
Alonzo  Clark  Scholar,  Assistant  in  Bacteriology  and  Hygiene, 

College  of  Physicians  and  Surgeons,  Columbia  University,  New  York. 

GENERAL  PART. 

introductory. 

The  foUowing  paper  contains  the  principal  details  and  results 
of  the  work  carried  on,  under  the  auspices  of  the  Medical  Com- 
mission for  the  Investigation  of  Acute  Respiratory  Diseases  of 
the  Department  of  Health  of  the  City  of  New  York,  at  the 
Bacteriological  Laboratory  of  the  College  of  Physicians  and 
Surgeons,  Columbia  University. 

In  mapping  out  the  bacteriological  work  of  the  Commission,  it 
was  deemed  advisable  to  institute  a  central  laboratory  to  which 
the  cultures  of  pneumococci  and  allied  organisms — ^particularly 
such  cultures  as  seemed  to  show  variations  from  the  classic 
pneumococcus  type — might  be  sent  by  the  various  workers  for 
further  study  and  identification.  Such  an  arrangement,  ad- 
mitting of  the  study  of  the  organisms  under  uniform  cultiu^al 
conditions  and  by  one  investigator  or  corps  of  investigators, 
would,  it  was  hoped,  not  only  prove  a  valuable  biological  study 
in  itself,  but  possibly  minimize  errors  of  identification  and  thus 
increase  the  value  of  statistics  ultimately  to  be  based  upon  the 
findings  in  the  series  of  cases  investigated. 

This  Ys^ork  was  undertaken  by  the  writer,  with  the  assistance 
of  Dr.  J.  H.  Borden  and  Dr.  C.  B.  Knapp.  The  practical  agglu- 
tination work  was  largely  carried  on  b}^  Dr.  Borden,  and  the 
morphological  and  culture  tests  by  Dr.  Knapp. 

145 


146       Comparative  Study  of  Pneumococci  and  Allied  Organisms 

For  the  information  of  the  various  workers  under  the  Com- 
mission, and  to  obtain  uniformity  in  the  scheme  of  investigation, 
a  comparatively  full  summary  of  the  characters  of  pneumococci 
and  streptococci,  and  the  chief  points  to  be  determined  in  their 
recognition  and  differentiation,  as  well  as  suggestions  as  to  the 
best  methods  of  isolating  them  in  pure  culture  and  preserving 
their  vitality,  was  sent  from  the  central  laboratory  to  each  inde- 
pendent worker.     The  workers  were  also  furnished  with  blanks  ^ : 

1  INFORMATION  TO  BE  OBTAINED  ABOUT  PERSONS  FROM  WHOM 
SPECIMENS  ARE  TAKEN.* 

Case  No Date: 

Age: Sex: Race: 

Residence:  City Country 

Occupation  (in  city  or  country) : 

Condition  of  Health: 

Present  State:    Condition    of    naso-pharynx,  mouth,  bronchiand  lungs,  and 
middle  ear? 

Past  History:    "Cold,"  catarrh,  laryngitis,  bronchitis,  or  any  inflammation 
with  which  pneumococcus  may  have  been  associated  ? 

Is  person  subject  to  "colds,"  bronchial  affections,  or  middle-ear  trouble?.  ... 


Does  person  use  alcohol  or  tobacco?      

Family  History:   Have  members  of  family  (mother,  father,  brother,  or  sister) 
suffered  from  an  attack  of  pneumonia  in  past  years? 

Association  with  Infected  Individuals: 

1.  Has,  or  has  had  recently,  any  member  of  immediate  family  a  "cold,"  catarrh, 

bronchitis,  pneumonia,  or  any  inflammatory  condition  with  which  pneumo- 
coccus may  be  associated  (middle-ear  trouble,  mastoiditis,  conjunctivitis)? 

2.  Has  person  otherwise  associated  or  come  into  contact  with  any  one  having 

a  "cold,"  bronchitis,  pneumonia,  or  middle-ear  inflammation,  mastoiditis, 
conjunctivitis? 

3.  Does  person  often  or  with  regularity  ride  in  crowded  tram,  elevated,  or 

steam  cars  ? 


4.  Does  person  frequent  theatres  or  crowded  shops  or  classrooms? . 


5.  Has  person  been  exposed  to  poisonous  gases  or  fumes,  or  other  deleterious 
aerial  agents  ? 

Subsequent  History:   Condition  of  person  1 5  days  subsequent  to  examination. 
Has  "cold,"  bronchitis,  or  pneumonia,  or  other  pneumococcus  infection  de- 
veloped ? 

Negative  Cases: 

Complete  clinical  records  should  always  be  kept  of  all  persons  from  whom 
specimens  for  examination  are  taken,  whether  organisms  be  obtained  or  not, 
in  order  to  determine  percentage  of  occurrence. 

(Signature) 

*  This  blank  is  to  be  filled  out,  and  sent  to  the  Central  Laboratory  with  the 
culture  and  its  description. 


Philip  Hanson  Hiss  147 

one  for  recording  the  data  concerning  individuals  from  whom 
cultures  were  taken,  and  the  other  for  recording  the  morphologi- 
cal, staining,  and  chief  cultural  characters  and  pathogenic  action 
of  the  organisms  isolated. 

These  blanks  were  to  be  filled  out  and  returned  with  the 
cultures,  for  the  information  of  the  workers  of  the  central 
laboratory.  2 

DESCRIPTION  OF  CHARACTERS  CULTURE.* 

Culture  No Case  No Date  of  isolation: 

Source  of  Culture: 

Saliva : Sputum : Throat  and  nose  swab : 

Technique  of  Isolation: 

By  Animal  Inoculation .  .  Rabbit  or  mouse  died  in days,    hrs. 

with  pure  or  mixed  infection. 

By  Culture  Method.     Agar  plate,  pour  or  streak 

Morphology  and  Staining:  (Gram's  stain.  Capsule  stain,  etc.) 

In  blood  or  exudate  of  original  mouse  or  rabbit 


From  culture  media,  agar,  coagulated  blood  serum , 

Were  similar  organisms  determined  in  smears  from  original  specimens? 

Cultural  Characters: 

Broth:    

Agar:   


Loeffler's  blood  serum:   

Gelatin:  Grows  at °  C.      (24°,  22°,  i8°  C.) 


Litmus  milk:  Acid  in days.     Coagulated  in days. 

Inulin  serum-water:   Coagulation  does  or  does  not  take  place  in days. 

Pathogenicity:   (Minimal  fatal  dose  of  suspension  of  24-hr.  agar  culture  in 

c.  c.  0.85  %  salt  solution). 

How  many  generations  removed  from  original  isolation  and  on  what  media? 

Rabbit,  weight gms.,  dies  in days,   hrs.,  from 

c.  c,  subcutaneously,  intravenously. 

Mouse  dies  in days, hrs.,  from c.  c.  subcutaneously. 

Agglutination: 

Agglutinates  at  i  to   in  immune    serum.     Control  test  with 

homologous  culture  shows  an  agglutination  at  i  to (Duration 

of  observation,  3,  12,  24  hrs.) 

Agglutinates  in  serum  of  person  from  whom  culture  was  isolated  at  i  to 

Other  observations  and  remarks: 

(Signature) 

*  This  blank  is  to  be  filled  out,  and  sent  with  cultures  to  Central  Laboratory. 

2  It  should  be  noted  here  that  the  work  of  each  independent  investigator, 
as  recorded  in  his  report  to  the  Commission,  represents  his  own  findings  and 
bacteriological  identifications  and  not  those  of  the  workers  of  the  central  lab- 


148       Comparative  Study  of  Pneumococci  and  Allied  Orgatiisms 

Object  of  the  Bacteriological  Study  Instituted  by  the  Commission. 
— As  biological  knowledge  widens  and  new  technical  methods 
are  discovered,  confidence  in  identifications  based  on  less  com- 
plete studies  and  more  primitive  methods  is  necessarily  shaken 
and  statistics  founded  on  these  lose  their  value.  It  is  only  with 
a  re-establishing  of  the  identity  of  organisms  in  conformity  with 
the  new  requirements,  or  by  a  determination  of  their  dissimilarity, 
that  the  old  statistics  can  again  assume  importance  or  may  be 
definitely  discarded. 

This  being  the  case,  although  much  work  had  already  been 
done  by  many  investigators  in  connection  with  the  bacterial 
flora  of  the  normal  mouth,  and  although  no  reasonable  doubt 
existed  as  to  the  presence  of  the  pneumococci  in  the  mouths  of 
a  certain  percentage  of  healthy  individuals,  still  it  was  thought 
advisable  to  confirm,  if  possible,  and,  it  might  be,  extend  these 
statistics  so  far  as  practicable  in  the  light  of  recent  developments 
in  our  knowdedge  of  the  biology  of  pneumococci  and  strepto- 
cocci, and  to  apply  to  the  recognition  and  identification  of  these 
organisms  the  newer  biological  and  technical  methods.  By 
such  a  study  it  seemed  fair  to  suppose  the  true  nature  of  the 
organisms  from  the  mouths  of  healthy  persons  could  be  deter- 
mined, and  that  their  identity  or  non-identity  with  pneumococci 
derived  from  persons  suffering  from  pneumonia  and  various 
pneumococcic  infections  could  be  definitely  established. 

Source  of  Cultures  Examined  .—The  scope  of  the  investigation 
included  not  only  the  examination  of  cultures  isolated  from  the 
saliva  and  naso-pharynx  of  presumably  healthy  individuals,  but 
also  of  organisms  obtained  from  individuals  suffering  from 
"colds,"  pharyngitis,  and  bronchitis. 

Cultures  isolated  from  the  sputum  of  pneumonia  patients,  or 
from  their  lungs  at  autopsy,  as  well  as  cultures  from  more  truly 

oratory.  No  systematic  reports  have  been  sent  by  the  central  laboratory  to 
those  sending  cultures,  and  their  reports  have  not  therefore  been  influenced  by 
the  work  of  the  central  laboratory.  Such  a  plan  has  the  advantage  of  show- 
ing the  percentage  of  discrepancy  between  the  identifications  of  the  same  or- 
ganisms by  different  workers,  and  the  disadvantage,  in  connection  with  the 
present  series  of  independent  papers,  in  the  interpretation  of  statistics  based 
upon  the  findings. 


Philip  Hanson  Hiss  149 

internal  sources,  such  as  the  circulating  blood  of  pneumonia 
patients  and  other  septicaemic  cases,  pleuritic  exudates  (em- 
pyaema),  the  spinal  fluid  in  cerebro-spinal  meningitis,  abscesses, 
otitis  media,  or  joint  infections,  were  also  investigated,  so  as  to 
afford  an  extensive  basis  of  comparison,  and  to  determine  the 
variations  in  morphology  and  biological  characters  that  might 
occur  among  organisms  of  the  pneumococcus  type,  which  were 
found  associated  with  pathologic  lesions  and  definite  infections. 

Plan  of  Biological  Study. — In  undertaking  the  investigation  of 
such  an  extensive  series  of  organisms,  it  was  realized  that  only 
the  most  promising  lines  of  study  could  be  followed  in  extenso 
at  the  central  laboratory,  and  that  many  suggested  methods  of 
differentiation,  as  well  as  a  detailed  study  of  pathogenicity  and 
the  pathogenic  effects  could  not  be  profitably  undertaken.  It 
was  hoped  that  these  studies  would  be  carried  out  in  detail  by 
the  other  workers  under  the  Commission,  and  the  value  of  such 
suggested  differential  methods  and  pathogenic  characters  as  a 
means  of  classification  be  thus  determined. 

The  work  of  this  laboratory  has  been  chiefly  confined  to  the 
determination  of  morphological  characters  and  staining  re- 
actions, and  of  the  growth  characters  presented  by  the  organism 
on  or  in  the  usual  culture  media,  and  their  fermentative  activities 
in  the  presence  of  various  carbohydrates  and  especially  of  inulin. 

The  immunization  of  animals  against  different  strains  of  or- 
ganisms and  the  careful  study  of  the  agglutinating  action  of 
the  sera  of  such  animals  on  the  various  organisms  were  also  given 
much  attention,  and  some  of  the  most  valuable  results  reached 
have  been  arrived  at  through  these  studies  in  agglutination. 

SPECIAL  PART. 

The  great  number  of  organisms  studied — in  all  about  260 — 
precludes  a  detailed  description  of  any  but  those  representing  the 
most  important  types  or  variations  from  these  types.  The  most 
important  details  from  the  standpoint  of  differentiation  and 
classification,  however,  are  given  in  outline  in  a  table  at  the  end 
of  the  report. 


150         Comparative  Study  of  Pneumococci  and  Allied  Organisms 

MORPHOLOGY. 

The  morphology  of  the  pneumococcus  is  in  general — especially 
when  repeated  examinations  are  made — one  of  the  most  valu- 
able guides  to  its  identity. 

When  typical  the  pneumococcus  is  a  rather  large,  lancet- 
shaped  coccus  occurring  in  pairs  and  surrounded  by  a  definite 
and  often  wide  capsule,  which  usually  includes  the  two  approxi- 
mated cocci  without  a  definite  indentation  opposite  their  line  of 
division.  The  pneumococci  may,  however,  occur  singly,  or  in 
short  chains,  and  even  fairly  long  chains  are  not  infrequently 
met  with  imder  artificial  culture  conditions.  These  may  be  chiefly 
due  to  the  cultural  conditions  or  be  a  prominent  characteristic 
of  certain  strains.  Apparently  the  capsules  of  organisms  making 
up  the  chains  are  continuous;  wavy  indentations  are  usually 
present,  however,  in  the  capsule  of  chains,  and  at  times  distinct 
divisions  are  observed. 

The  chief  variations  from  the  typical  morphology  consist  either 
in  the  assumption  of  a  more  distinctly  spherical  coccus  type,  or 
in  an  elongation  approximating  the  bacillary  form.  Under  cer- 
tain conditions  of  artificial  cultivation  a  distinct  flattening  of  the 
organisms,  particularly  those  making  up  chains,  may  be  seen, 
and  even  the  impression  of  the  existence  of  a  longitudinal  line  of 
division,  characteristic  of  many  streptococcus  cultures,  is  not  in- 
frequently gained. 

The  capsules  imder  certain  conditions,  especially  in  artificial 
media,  may  be  absent  or  not  demonstrable,  and  in  certain  strains 
capsules  apparently  may  not  be  present  tmder  any  conditions. 

It  is  readily  seen,  therefore,  that  when  the  capsule  is  absent 
and  the  pneumococcus  has  at  the  same  time  asstimed  the  spherical 
or  flattened  streptococcus  type,  identification  by  morphological 
examination  is  not  possible.  Even  if  the  capsules  are  still  intact, 
a  definite  identification  of  such  streptococcus-like  forms  is  not 
permissible,  since  encapsulated  streptococci  are  not  infrequently 
met  with,  which  by  cultural,  fermentation,  and  agglutination 
tests  are  definitely  separable  from  pneumococci. ^ 

3  See  Hiss,  CM.  f.  Bakt.,  1902,  xxxi,  302,  and  Jour.  Exp.  Med.,  1905,  vii,  317. 


Philip  Hanson  Hiss         '  151 

In  the  opinion  of  the  writer  (Hiss),  no  classification  based 
solely  on  morphology  and  the  presence  or  absence  of  capsules, 
or  the  finer  morphological  variations  of  capsule  formation,  is  of 
value.  Practically  any  of  the  described  variations  may  domi- 
nate one  and  the  same  culture  under  different  or  even  ap- 
parently the  same  conditions  of  cultivation,  and  all  grades 
may  occur  in  capsule  development  from  its  typical  formation, 
through  all  variations,  to  its  total  and  apparently  permanent 
absence. 

Streptococci,  as  has  been  noted,  may  show  capsule  formation. 
This  usually  occurs,  so  as  to  be  demonstrable  by  current  capsule 
staining  methods,  only  under  certain  and  not  well  understood 
conditions,  and  the  organisms  tend  to  lose  this  character  much 
more  readily  under  artificial  conditions  than  do  the  pnemno- 
cocci.  While  some  strains  retain  their  power  of  forming  capsules 
through  many  generations,  the  majority,  under  ordinary  cultural 
conditions,  usually  have  no  demonstrable  capsule. 

Conditions  Favoring  Capsule  Development  and  Demonstration. 
— The  most  favorable  conditions  known  for  the  development  of 
the  pneumococcus  capsules  are  found  in  the  body  fluids  of  man 
and  animals  suffering  from  pneumococcus  infection.  For  in- 
stance, capsules  may  be  demonstrated  with  ease  by  the  usual 
methods  in  the  blood,  serum,  and  inflammatory  exudates  of  the 
infected  rabbit  and  white  mouse,  which  are  among  test  animals 
the  most  favorable  for  these  experiments.  Capsules  may  be 
equally  well-marked  in  the  fresh  sputum  of  pneumonia  patients, 
especially  in  the  early  stages  of  the  disease,  and  in  the  exudates 
accompanying  such  pneumococcus  infections  as  meningitis, 
otitis  media,  and  empysema.  In  sputum  and  the  exudates  of  the 
various  localized  infections,  the  organisms  are,  however,  fre- 
quently degenerated  or  under  chemical  conditions  unfavorable 
for  capsule  staining,  and  satisfactory  results  are  not  then  easily 
to  be  obtained. 

The  same  is  true  of  the  scrapings  from  lungs  of  patients  dead 
of  pneumonia,  often  even  in  the  state  of  red  hepatization. 

In  artificial  cultivation,  if  the  nutrient  medium  is  not  milk 
or   does    not    contain     serum    (serum,    serum-agar,     Loeffier's 


152         Comparative  Study  of  Pneumococci  and  Allied  Organisms 

coagtilated  blood-serum),  capsules,  by  the  ordinary  methods  of 
preparing  and  staining,  are  not  usually  to  be  demonstrated. 

Capsules  may,  however,  with  much  regularity  be  demon- 
strated on  pneumococci  from  agar,  broth,  or  almost  if  not  all 
artificial  media,  irrespective  of  the  length  of  time  the  organisms 
have  been  under  artificial  cultivation,  if  beef  or  rabbit  serum  is 
used  as  the  diluent  when  they  are  spread  on  the  cover-glass  for 
staining.  The  copper  sulphate  or  potassium  carbonate  method 
will  then  stain  them  readily.* 

Capsules  are  usually  not  so  readily  demonstrated  on  strepto- 
cocci, no  matter  what  their  source,  and  with  no  regularity,  as 
compared  with  pneumococci.  They  may,  however,  often  be  de- 
monstrated by  the  use  of  the  potassium  carbonate  method,  when 
the  organisms  have  been  grown  in  sugar-serum  media  or  on 
ascitic-agar  and  then  smeared  on  the  cover-glass  with  serum.  In 
some  ctiltures  the  capsules  are  quite  as  sharp  and  well-defined  as 
those  of  pneumococci,  in  others  they  are  less  well-marked,  and 
in  some  appear  as  if  in  a  semi-solid  state  and  on  the  point  of 
dissolving.  Whether  the  capsules  noticed  on  certain  strepto- 
cocci are  of  the  same  nature  as  that  of  the  pneumococcus  is  at 
present  open  to  doubt. 

GENERAL    CULTURAL    CHARACTERS. 

The  growth  characters  on  the  usual  culture  media,  as  was 
to  be  supposed  from  our  own  previous  work  and  the  publica- 
tions of  other  investigators,  have  afforded  no  practical  basis  for 
the  differentiation  of  pneumococci  from  various  simulating  or- 
ganisms and  from  streptococci.  Routine  tests  were,  however, 
made  in  neutral  infusion-broth  and  gelatin,  and  on  neutral  in- 
fusion-agar  and  Loeffler's  coagulated  blood-serum,  and  in  litmus 
milk.  With  the  exception  of  gelatin  and  Loeffler's  sertim,  no 
constant  characters  of  diagnostic  and  differential  worth  were 
developed. 

On  Loeffler's  coagulated  blood-serum  the  pneumococcus  usu- 
ally develops  into  moist,  rather  watery  discrete  colonies  which 

*  See  Hiss,  ref.  cit. 


Philip  Hanson  Hiss  153 

tend  to  disappear  after  some  days,  while  the  streptococcus 
colonies,  though  also  discrete,  are  usually  drier  and  slightly 
whiter  in  appearance  than  those  of  the  pneumococcus.  The 
colonies  of  these  two  organisms,  however,  often  so  closely  simu- 
late each  other  that  they  cannot  definitely  be  distinguished. 
The  Loeffler  serum  has,  however,  been  of  value  in  aiding  in  the 
separation  of  the  so-called  Streptococcus  mucosus  from  pneumo- 
cocci.  Streptococcus  mucosus  grows  on  this  medium  in  a  defi- 
nite, smooth,  watery  layer,  with  fairly  even  edges,  while  the 
pneumococcus,  as  above  noted,  usually  occurs  in  discrete  watery 
colonies ;  and  even  when  these  colonies  coalesce,  there  is  not  the 
same  smoothness  to  the  surface  and  edges  of  the  growth  as  is 
shown  by  that  of  Streptococcus  mucosus. 

Gelatin  has  been  useful  in  determining  the  limit  of  low-tem- 
perature growth  of  the  cultures,  and  in  identifying  Streptococcus 
mucosus  cultures. 

Many  cultures  of  pneumococci  will  not  develop  in  gelatin  at 
temperatures  lower  than  22°  C.  Others,  however,  even  when 
freshly  isolated,  will  grow  fairly  abundantly  in  gelatin  at  this 
temperature.  This  is  true  not  only  of  typical  organisms  from 
pneumonic  sputum  and  other  pathologic  sources,  but  equally  so 
of  organisms  from  the  mouths  of  healthy  persons,  so  that  in  these 
peculiarities  of  growth  or  non-growth  at  22°  C.  or  below,  the 
mouth  organisms  have  been  found  to  agree,  and  in  about  the 
same  proportion,  with  pneumococci  from  pathologic  sources. 

Streptococci  and  other  organisms  from  the  mouth,  which  are, 
so  far  as  determinable  at  present,  not  pneumococci,  usually  grow 
readily  and  abundantly  in  gelatin  at  22°  C.  or  below.  As  their 
growth  is  generally  closely  similar  to  that  of  the  pneumococcus 
in  gelatin,  this  test  is  of  little  value  in  differential  diagnosis.  In 
the  case,  however,  of  Streptococcus  mucosus  the  growth  along 
the  puncture  in  neutral  infusion-gelatin,  after  some  days'  de- 
velopment at  ordinary  room  temperatures,  is  readily  to  be  dis- 
tinguished from  that  of  the  pneumococcus  and  Streptococcus 
pyogenes.  Instead  of  being  a  growth  made  up  of  fine  discrete 
colonies  representing  the  original  line  of  inoculation,  as  in  the  case 
of  pneumococci  and  of  streptococci  of  various  descriptions,  the 


154:        Comparative  Study  of  PneuTnococci  and  Allied  Organisms 

growth  of  Streptococcus  mucosus,  especially  in  the  deeper  por- 
tions, where  it  seems  to  develop  most  abundantly  and  character- 
istically, is  made  up  of  larger  globe-like  colonies,  which  give  one 
the  impression  that  slight  fiuidification  of  the  gelatin  has  taken 
place.  This  appearance  is  probably  due  to  the  large  amoiuit  of 
capsular  substance  developed  by  this  organism  and  is  one  of  its 
most  constant  and  distinctive  characteristics.  Cultures  of  this 
organism  obtained  by  us  from  various  sources,  in  the  early  days 
of  our  work,  have  continued  to  display  this  character  and  their 
typical  growth  on  Loeffler's  or  other  serum  media,  without  the 
least  noticeable  change.  This  indicates  that,  no  matter  what 
the  evolution  and  relationships  of  this  organism  may  have  been 
or  are,  the  characters  now  distinguishing  it  from  pneumococci 
are  certainly  in  most  instances  of  a  markedly  constant  character, 
and  that  it  is  not  apt  rapidly  to  assume  the  characters  of  typical 
pneumococci  on  the  one  hand,  or  streptococci  on  the  other.  Ob- 
servations indicating  such  a  change  may  possibly  be  based  on  a 
mistake  in  primarily  identifying  a  more  or  less  confluent  Loeffler's 
serum  growth  of  a  true  pneumococcus  with  that  of  Streptococcus 
mucosus. 

FERMENTATION    TESTS ACID    PRODUCTION. 

When  we  turn  from  the  cultural  characters  as  developed  on 
and  in  the  usual  culture  media — which  are  of  so  little  aid  in 
differentiating  pneumococci  from  streptococci — to  certain  of  the 
physiologic  processes  of  these  organisms,  as  indicated  by  their 
action  on  carbohydrates,  the  field  of  investigation  is  widened  and 
decidedly  more  promising.  In  a  series  of  investigations  carried 
on  by  one  of  us  (i)  some  four  years  ago,  but  only  recently  pub- 
lished in  detail,  it  was  shown  that  pneumococci  have  marked 
fermentative  abilities,  and  that  these  are  of  wider  scope  than 
those  possessed  by  the  typical  Streptococcus  pyogenes  and  prob- 
ably many  other  unclassified  streptococci. 

Pneumococci,  it  was  shown,  produced  acid  with  ease  from 
monosaccharids,  disaccharids,  and  such  complex  saccharids  as 
dextrin,  glycogen,  starch,  and  inulin.  Streptococci,  on  the  other 
hand,  as  represented  by  various  supposed  strains  of  the  pyogenes 


Philip  Hanson  Hiss  155 

type,  although  producing  acid  with  much  regularity  from  some 
of  these  carbohydrates,  usually  did  not  have  the  ability  to  fer- 
ment starch  and  glycogen  with  the  same  regularity  and  ease  as 
the  pneumococci,  and  never,  so  far  as  the  observations  on  about 
fifty  cultures  indicated,  were  able  to  produce  acid  from  inulin. 
Inulin  fermentation  was  therefore  looked  upon  as  a  definite 
character  of  pneumococci,  and  probably  as  a  valuable  aid  in 
differentiating  atypical  pneumococci  from  the  non-inulin- 
fermenting  Streptococcus  pyogenes.  Whether  other  species  of 
streptococci  or  other  lancet-shaped  organisms  simulating  pneu- 
mococci possessed  such  an  inulin-fermenting  ability  was  not  at 
that  time  determined. 

The  medium  used  as  a  basis  for  these  fermentation  tests  was 
serum-water,  composed  of  beef-serum  one  part  and  distilled 
water  two  or  three  parts,  to  which  one  per  cent,  of  a  five  per 
cent,  solution  of  highly  purified  litmus  was  added.  The  medium 
was  then  heated  to  ioo°  C.  for  a  few  moments  and  the  various 
carbohydrates  added  in  the  proportion  of  one  per  cent.,  after 
which  it  was  sterilized  in  the  regular  manner  on  three  consecutive 
days. 

In  the  present  investigation,  this  same  medium  has  been  used 
as  the  nutrient  base  in  the  fermentation  tests,  which  have  in- 
cluded a  study  of  all  the  organisms  in  dextrose,  lactose,  maltose, 
saccharose,  raffinose,  dextrin,  glycogen,  and  inulin,  as  well  as  the 
alcohols  mannit  and  dulcit. 

A  careful  testing  of  the  organisms  sent  to  us  by  the  various 
workers,  as  well  as  those  isolated  by  ourselves  during  this  work, 
has  again  demonstrated  the  inulin-fermenting  power  of  pneu- 
mococci, and  the  value  of  this  simple  test  in  separating  them 
from  typical  pyogenic  streptococci  as  well  as  from  various  defi- 
nitely encapsulated  cocci  which  closely  simulate  the  true  pneu- 
mococcus.  On  the  other  hand,  certain  inulin-fermenting 
organisms  have  come  to  hand  which  we  have  not  so  far  been 
able  satisfactorily  to  identify  either  as  pneumococci  or  strepto- 
cocci of  the  pyogenes  type.  Some  of  these  are  definitely  neither 
pneumococci  nor  streptococci  of  well-marked  type,  while  others 
have  a  definite  pneumococcus  morphology  but  no  capsule,  or  are 


156         Comparative  Study  of  Pneumococci  and  Allied  Organisms 

typically  streptococci,  the  members  of  the  latter  being  in  general 
spherical  and  often  occurring  in  long  chains.  These  organisms 
for  the  most  part  lack  capsules  or  only  possess  poorly  developed 
ones.  It  is  worthy  of  note  that  most  of  these  atypical  organisms 
were  isolated  either  from  the  circulating  blood  of  patients,  or 
from  some  other  internal  source,  or  in  many  cases  from  pneu- 
monic or  other  lungs  at  autopsy.  The  question  of  their  probable 
identity  will  be  considered  more  in  detail  in  connection  with  the 
results  obtained  by  agglutination  tests  in  immune  sera. 

The  chief  point  brought  out  by  these  fermentations,  so  far  as 
its  bearing  on  the  immediate  problem  before  the  Commission  is 
concerned,  is  that  organisms  morphologically  of  the  pneumo- 
coccic  type  from  the  mouths  of  healthy  individuals  correspond 
absolutely  in  their  fermentative  characters  with  typical  pneu- 
mococci from  other  sources,  and  thus  a  definite  link  is  added  to 
the  chain  of  their  connection  with  true  pneumococci. 

In  studying  organisms  of  the  same  or  closely  similar  mor- 
phology, particularly  such  organisms  as  cannot  be  separated 
from  each  other  by  morphological  or  cultural  characters  of 
diagnostic  value,  two  methods  of  great  value  have  been  de- 
veloped during  late  years — fermentation  tests,  and  the  test  for 
agglutination  in  immune  sera.  By  these  two  methods  differences 
in  organisms  have  been  discerned  that  were  hardly  suspected,  or 
only  guessed  at  by  earlier  investigators.  In  using  such  methods, 
however,  which  deal  with  the  truly  physiologic  activities  of 
organisms,  care  and  patience  must  be  expended  upon  prolonged 
observations  and  repeated  tests.  Due  regard  must  be  paid  to  the 
fact  that  transient  and,  it  may  be,  permanent  modifications 
may  occur  in  the  physiologic  functions  of  organisms  by  exposure 
to  unusual  or  adverse  environment,  and  observations,  therefore, 
based  on  short  or  superficial  studies  or  tests  of  such  organisms 
may  be  misleading.  These  changes  are  usually  to  be  noted  in 
the  suppression  or  weakening  of  functions,  upon  which  fer- 
mentations, agglutinations,  and  pathogenicity  depend,  and 
hardly  extend  to  the  acquisition  of  totally  new  functions,  which 
probably  are  the  product  of  a  much  more  gradual  and  prolonged 
process   of   evolution   and   adaptation.     These   facts   are   well- 


Philip  Hanson  Hiss  157 

known,  but  attention  is  called  to  them  here  in  connection  with 
the  interpretation  of  fermentation  and  agglutination  results 
which,  unless  considered  from  this  broad  standpoint,  are  often 
confusing  or  lead  to  false  conclusions.  As  an  example,  we  may- 
use  the  variations  in  the  rate  of  fermentation  of  inulin  by  various 
cultures  of  pneumococci.  A  culture  in  full  possession  of  this 
faculty  may  produce  marked  acid  within  from  eighteen  to  twenty 
hours,  while  another  ctdture,  if  tested  only  over  five  or  six  days, 
might  be  thought  not  to  ferment  inulin;  after  a  longer  time,  or 
in  repeated  tests,  however,  this  function  will,  so  far  as  our  ex- 
perience goes,  eventually  reveal  itself.  Variations  in  the  rate  of 
fermentation  are  often  marked  in  the  same  culture,  differences  of 
days  in  the  time  required  for  complete  fermentation  often  being 
noted. 

In  the  absence  of  all  information  as  to  the  reaction  of  organ- 
isms in  specific  immune  sera,  fermentations  seem,  therefore,  the 
safest  guide,  other  things  being  equal,  to  the  identification  of 
organisms.  Where  fermentations  have  been  carefully  studied 
and  the  identity  of  organisms  thus  determined,  one  can  usually 
predict  with  much  certainty  what  the  results  of  tests  in  immune 
sera  will  be.  However,  it  must  be  remembered  that  variations 
in  agglutinative  functions  also  occur  and  may  be  fairly  per- 
manent, thus  leading  to  false  conclusions,  unless,  on  the  other 
hand,  due  regard  be  paid  to  the  other  biological  characters  as 
means  of  identification,  and  to  the  source  of  the  culture.  Changes 
in  the  limit  of  agglutination  of  undoubtedly  the  same  species  or 
even  the  same  culture,  after  exposure  to  certain  environments, 
are  well  known  to  those  familiar  with  typhoid  agglutinations, 
and  in  considering,  in  the-  following  section,  some  of  our  agglu- 
tination results,  the  same  phenomenon  will  be  noticed  to  occur 
among  the  pneumococci. 

AGGLUTINATION    TESTS. ^ 

In  undertaking  the  systematic  comparative  study  of  the 
agglutinations  of  pneumococci  and  allied  organisms  from  various 

s  The  table  has  been  placed  at  the  end  of  the  paper. 


158         Comparative  Study  of  Pneumococci  and  Allied  Organisms 

sources,  the  workers  under  the  Commission  had  to  enter  a 
practically  uncultivated  field  of  research.  No  guiding  statistics 
of  value  existed  and  the  technical  procedures,  at  least  those 
available  for  such  an  extensive  study,  had  not  been  worked  out. 
The  same  statement  is  equally  true  of  streptococcus  agglutina- 
tions and  agglutination  technique.  Agglutination  tests  with 
both  pneumococci  and  streptococci,  where  the  usual  broth 
cultures  (either  with  or  without  sugar  added)  or  emulsions  from 
agar  had  been  employed,  had  in  the  experience  of  most  workers 
been  found  not  only  technically  unsatisfactory,  but  had  given 
varying  and  often  contradictory  results. 

After  a  few  trials  of  the  older  methods  it  was  obvious,  there- 
fore, if  advances  were  to  be  made  in  our  knowledge  of  the  agglu- 
tinations of  these  organisms,  that  a  new  as  well  as  a  simple  and 
easily  handled  technique  had,  if  possible,  to  be  devised. 

The  first  problem  was  to  get  the  organisms  in  a  proper  and 
dense  enough  emulsion  for  observation  of  agglutination,  and  the 
second  to  obtain  them  in  the  biological  state  in  which  they 
would  respond  definitely  and  with  regularity  to  the  action  of 
the  agglutinins,  for  with  the  old  procedures,  as  stated,  only 
irregular  and  unsatisfactory  agglutinations  had  been  obtained. 
The  only  techinque  already  in  use  which  in  any  degree  con- 
formed to  these  conditions  was  that  recommended  by  Wads- 
worth.^  This  is  valuable  and  reliable,  but  when  a  long  series  of 
organisms,  as  in  this  comparative  study,  have  to  be  tested 
against  the  same  or  various  sera,  almost  a  prohibitive  amount  of 
time  and  energy  must  be  consumed,  when  following  this  tech- 
nique, in  centrifugating  and  preparing  the  organisms  for  the  tests. 

A  method  which  met  the  requirements  was  finally  worked  out 
by  us.  For  purposes  of  reference  it  is  given  here  in  the  words 
of  the  original  paper  which  has  but  recently  been  published  (2) : 

"The  method  gives  results  and  appearances  which  are  entirely  comparable 
to  those  familiar  in  typhoid,  dysentery,  and  various  other  agglutinations,  and 
is  simple  and  particularly  available  for  the  study  of  pneumococci  and  strepto- 
cocci and  other  organisms  which  ferment  carbohydrates,  but  which  thrive 
poorly  or  die  out  rapidly  in  the  usual  media.     The  streptococci  often,  as  is  well 

'Wadsworth,  Journ.  Med.  Research,  1903,  x,  228. 


Philip  Hanson  Hiss  .  159 

known,  grow  in  broth,  either  with  or  without  sugar,  in  floccuH  or  thickly  matted 
masses  entirely  useless  for  agglutination  purposes.  By  the  proposed  method 
usually  a  fair  and  often  a  good  and  satisfactory  emulsion  may  be  obtained, 
from  which  agglutinative  limits  may  easily  be  determined. 

"The  medium  used  should  be  a  one-  or  two-per-cent.  peptone  broth  made 
from  meat  infusion,  which  has  been  brought  to  neutrality  before  boiling  and 
coagulation.  After  filtration  for  clearing,  one  per  cent,  of  dextrose  (or  other 
sugar  fermentable  by  the  given  organism)  and  one  per  cent,  of  calcium  car- 
bonate are  added.  If  the  medium  be  acid,  the  latter  salt  will  of  course  bring  it 
to  neutral.  The  calcium  carbonate  may  then  be  well  distributed  throughout 
the  broth  by  shaking  and  the  emulsion  rapidly  decanted  into  tubes  or  preferably 
small  Erlenmeyer  or  Florence  flasks,  say  loo  cubic  centimeters  to  150  cubic 
centimeters  in  each.  These  are  sterilized  on  three  consecutive  days  at  100°  C. 
in  the  usual  manner.  The  flasks  after  inoculation  are  placed  at  37°  C.  and  are 
thoroughly  shaken  once  or  twice  a  day  to  neutralize  the  acid  formed  and  to 
break  up  the  chains  and  masses  in  the  case  of  streptococci.  The  growth  may 
be  sufficient  for  purposes  of  agglutination  in  two  days  or  even  in  one  day, 
but  as  a  routine  up  to  the  present  time  we  have  employed  a  three  or  four 
days'  growth,  which  seems  to  give  more  uniform  results  and  more  marked 
agglutination. ' 

"About  an  hour  before  using  for  agglutination  tests  the  culture  should  be 
thoroughly  shaken  and  the  calcium  carbonate  and  larger  clumps,  if  present, 
allowed  to  settle  dxiring  this  time.  The  sample  to  be  tested  should  then  be 
taken  from  the  upper  portion  of  the  fluid;  or  the  cultures,  after  shaking,  may 
be  centrifugated  for  a  few  minutes.  This  centrifugalization,  however,  is  not 
necessary  if  one  remembers  that  a  slight  primary  deposit  may  occur  which  is 
not  due  to  agglutination. 

"  Routine  agglutination  tests  are  made  by  adding  one  cubic  centimeter  of  the 
serum  dilution  to  one  cubic  centimeter  of  the  emulsion  in  small  test-tubes.  The 
tubes  are  placed  at  3  7°  C.  for  two  or  three  hoiirs — after  which  time  the  agglutina- 
tions are  often  practically  complete — and  then  transferred  to  the  ice-box  to 
prevent  growth  taking  place  and  permit  of  the  ftirther  deposition  of  the  clumps 
of  agglutinated  organisms,  and  the  final  control  reading  made  after  eighteen 
to  twenty-four  hours. 

"Spontaneously  agglutinating  cultures,  of  course,  are  met  with  among  the 
pneumococci,  and  are  frequent  with  streptococci,  but  even  with  these  the  limits 
of  agglutination  can  be  determined  with  much  certainty  if  careful  comparison 
with  the  control  is  made." 

In  the  course  of  our  work  many  hundreds  of  serial  tests  have 
been  made  by  this  method,  and  it  has  proved  of  great  value  not 
only  in  the  ease  of  application  but  in  the  comparative  uniformity 
of  results  given  by  it.     Readings  up  to  eight  hundred  and  over 

'  Such  cultiu-es  may  then  be  preserved  in  the  ice-box.  We  have  tested  them 
frequently  in  the  same  sera  from  day  to  day,  and  have  found  little  or  no  change 
in  their  limit  of  agglutination  even  after  weeks. 


160         Comparative  Study  of  Pneumococci  and  Allied  Organisms 

are  not  rare  in  the  sera  of  rabbits  immunized  to  pneumococci, 
and  the  serum  of  streptococcus  immune  rabbits  has  in  some 
instances  agglutinated  the  homologous  streptococcus  cultures 
in  dilutions  over  6400.  Reported  agglutinations  of  pneumo- 
cocci made  by  the  usual  method  rarely  indicate  an  agglutinating 
power  of  the  sera  over  100,  and  such  readings  are  obtained  with 
no  uniformity.  Our  success  in  getting  such  high  agglutinations 
has  probably  not  only  been  due  to  our  method  of  making  the 
test,  but  in  part,  at  least,  to  the  use  for  inoculation  of  the  ani- 
mals of  mass  cultures  obtained  by  growing  the  organism  for  this 
purpose  in  the  calcium-carbonate-glucose  broth.  Our  animals, 
although  usually  very  gradually  immunized  at  first,  eventually 
received  large  doses  of  these  growths  intravenously,  and  for  the 
most  part  survived  them  and  remained  in  good  condition. 
Rabbits,  however,  immunized  against  Streptococcus  mucosus 
in  several  instances  became  much  emaciated  and  eventually  died. 

A  series  of  animals  was  immunized  with  pneumococci  from 
pneumonic  sputum,  and  from  the  mouths  of  healthy  individuals, 
also  with  Streptococcus  pyogenes,  Streptococcus  mucosus,  and 
various  other  organisms  of  peculiar  types.  The  sera  obtained 
from  these  animals  and  from  normal  rabbits  afforded  the  basis 
for  an  extended  study  of  the  agglutination  reactions  of  the 
various  cultures. 

All  the  organisms  of  our  series  have  been  tested  against  the 
immune  pneumococcus  sera,  and  their  agglutinations  controlled 
by  tests  in  normal  rabbit  serum  and  normal  salt  solution,  and  a 
careful  comparison  made  with  the  results  given  by  the  homolo- 
gous immiinizing  cultures. ^ 

Pneumococci  as  a  rule,  no  matter  what  their  source,  do  not 
agglutinate  in  high  dilutions  of  normal  rabbit  serum,  rarely  over 
1:10  to  1 :  20,  nor  have  they  been  found  to  agglutinate  to  any 

8  There  may  be  a  slight  false  primary  clumping  in  all  tubes  of  the  series,  in- 
cluding the  controls.  These  clumps  are  apt  to  disappear  later.  Even  if  they 
persist  they  do  not  settle  out  rapidly,  and  the  specific  agglutination  is  readily 
distinguished  from  them.  Such  occurrences,  however,  should  make  one  ex- 
ceedingly cautious  in  accepting  results  reported  in  pneumococcus  and  strepto- 
coccus agglutinations  when  the  hanging-drop,  microscopic-test  method  alone 
has  been  employed. 


Philip  Haason  Hiss  '  161 

marked  extent  in  streptococcus  immune  serum  or,  with  the  ex- 
ception of  Streptococcus  mucosus  serum,  in  the  immune  serum 
obtained  by  inoculation  with  other  organisms  of  various  types. 

Nearly  without  exception,  however,  organisms  previously 
recognized  by  morphological,  staining,  and  fermentation  tests 
as  distinctly  of  the  pneumococcus  type,  have  been  found  to  ag- 
glutinate in  pneumococcus  immune  serum,  and  the  results  ob- 
tained, taking  the  series  of  these  organisms  as  a  whole,  have 
shown  remarkable  uniformity  in  degree  of  agglutination  and  a 
close  approximation  to  that  given  by  the  homologous  organism. 
This  is  true  not  only  of  pneumococci  from  recognized  pathologic 
sources,  such  as  pneumonic  sputum,  etc.,  but  of  the  organisms  of 
pneumococcic  type  from  the  mouths  of  supposedly  healthy  per- 
sons. The  same  results  are  obtained  in  the  sera  of  animals  im- 
munized against  these  latter  as  in  the  sera  of  those  immunized 
against  pneumococci  from  pathologic  sources.  In  other  words, 
the  agglutination  tests  have  fully  confirmed  the  complete  iden- 
tity, which  was  presaged  by  the  fermentation  tests,  of  organisms 
of  pneumococcic  type,  from  the  mouths  of  healthy  individuals, 
with  those  from  pathologic  sources.  Further  than  this,  these 
tests  have  also  reinforced  the  evidence  given  by  fermentation 
tests,  namely,  that  there  are  distinctly  encapsulated,  Gram- 
positive  organisms  which  may  be  met  with  and  which  simulate 
pneumococci  too  closely  for  morphological  separation,  but  which 
are,  nevertheless,  according  to  agglutination  and  fermentation 
tests,  separate  and  distinct  from  pneumococci.  These  remarks 
do  not  apply  to  Streptococcus  mucosus,  which  is  peculiar  in 
its  agglutination  reactions,  showing  only  moderate  agglutination 
in  the  sera  of  animals,  which  according  to  all  standards  are  highly 
immunized  against  it.  Agglutinations  of  i :  20  to  i :  50  generally 
abruptly  mark  the  limit.  In  pneumococcus  immune  serum  it 
shows  little  or  no  agglutination.  On  the  other  hand,  pneumo- 
cocci agglutinate  in  mucosus  immune  serum  in  very  high 
dilutions.  From  this  fact  and  the  fact  that  the  fermentations 
caused  by  these  two  organisms  are,  so  far  as  we  know,  coexten- 
sive, we  have  been  practically  forced  to  the  conclusion  that 
Streptococcus  mucosus  is  not  a  distinct  species,  but  a  variety  of 


162         Comparative  Study  of  Pneumococci  and  Allied  Organisms 

pneiimococcus,  which  is,  however,  very  firmly  established  in 
the  possession  of  certain  morphological  peculiarities,  especially  as 
relates  to  the  abundant  production  of  mucinous  or  capsular  ma- 
terial. Its  peculiarities  in  agglutination,  or  rather  non-agglu- 
tination, may  be,  we  have  thought,  closely  connected  with  the 
over-production  of  this  special  mucous  or  mucinous  material  and 
its  solution  in  the  culture  fluids — in  other  words,  that  this  material 
may  have  a  combining  or  inhibitive  action  on  the  agglutinins. 
In  cultures  centrifugated  and  washed  in  salt  solution  we  have, 
however,  failed  to  increase  markedly  its  agglutination,  though 
some  slight  increase  did  take  place.  What  the  explanation  is 
we  at  present  do  not  know,  but  the  fact  that  pneumococci  ag- 
glutinate to  such  a  marked  degree  in  Streptococcus  mucosus 
immune  serum  argues  a  close  relationship  between  the  two. 


Immune  Sera. 


Pneum. 


Pneum. 
"3." 


Pneum. 
"23." 


S.  muc. 
"7." 


S.  muc. 
"7-a." 


S.  pyog. 


Pn. 


S.  pyog. 
S.  muc. 


I    . . 

"3"-- 
"23".. 

'  4S"-- 
"E.  I 
"E.  32 
"E.  55 
"N.  7 
"_N.  17 
"  I  "  .  . 
'7"... 
'22"  . 
'30".. 

'55-  a' 
'Br.". 


100—400 
200—800 
200—800 


o— 10 
o— 10 


200—000 
400-800 
100—800 
400—800 
100—800 
200—800 


200—800 
o-io 
o— 10 
0—20 


400-800 

200-800 
100—200 


400—800 

800 
800 


200—000 

100—200 
200—800 

100  + 
200—400 
200—800 
200—800 

10-100 

10-50 

10—20 

10-50 
10-50 


400 
200—800 


100—400 
100—200 


200—800 
200—800 

10-200 
0-50 
0—20 


o-ioo 
o— 100 


800-6400 
0-50 


The  immunizing  organisms  Pn.  "i"  and  Pn.  "3"  are  from  pneumonic 
sputum:  Pn.  "23"  is  from  the  normal  mouth;  and  the  other  cultures  from 
normal  mouths  and  various  pathologic  soiirces. 

The  italics  indicate  the  agglutination  in  the  homologous  serum. 

When  two  numbers  are  used  to  record  the  results  of  a  test,  the  first  indicates 
the  last  dilution  in  which  full  precipitation  of  the  agglutinated  organisms 
occiirred,  and  the  second  the  highest  reading  with  a  hand  lens. 

The  results  of  control  tests  in  normal  rabbit  serum  are  not  recorded  in  the 
table.  A  few  of  the  pneumococcus  cultures  showed  a  very  slight  agglutination 
at  1:10.  The  Streptococcus  pyogenes  culture  agglutinated  at  1:100.  None 
of  the  Streptococcus  mucosus  cijltures  showed  agglutinations  at  1:10. 

The  above  table  illustrates  the  agglutination  of  pneumococci 


Philip  Hanson  Hiss  163 

in  pneumococcus  and  Streptococcus  mucosus  immune  sera, 
as  well  as  in  Streptococcus  pyogenes  immune  serum  and  normal 
rabbit  serum.  The  agglutinations  of  Streptococcus  mucosus 
and  Streptococcus  pyogenes  are  included  in  the  table  for  com- 
parison. 

Before  leaving  the  subject  of  the  agglutination  of  pneumo- 
cocci,  which  are  typical  morphologically  as  well  as  in  their  fer- 
mentation reaction,  it  must  be  noted  that  certain  of  them  when 
compared  with  the  homologous  organisms  in  immune  serum 
show  a  very  low  grade  of  agglutination.  A  glance  at  the  full 
table  at  the  end  of  the  report  will  show  that  such  organisms  are 
usually  not  from  the  normal  mouth  or  from  pneumonic  sputum, 
but  from  some  more  internal  source,  such  as  the  blood,  or  some 
chronic  and  deep-seated  lesion,  or  lungs  at  autopsy.  These  re- 
sults are  in  line  with  those  found  in  the  case  of  typhoid  bacilli 
from  the  circulation  and  from  more  chronic  lesions,  as  well  as 
those  grown  artificially  in  immune  serum,  and  are  probably  an 
illustration  of  the  modification  of  function  by  environment. 

Of  the  organisms  examined  by  us  which  do  not  ferment  inulin 
and  which  are  of  typical  streptococcus  morphology,  or  even  of 
diplococcus  type  and  slightly  lancet-shaped  but  non-capsu- 
lated,  little  need  be  said  here.  The  results  of  their  agglutination 
tests  will  be  found  in  the  appended  table.  It  is  simply  worthy 
of  note  that  some  streptococci  show  marked  agglutination  in 
normal  rabbit  serum  and  naturally,  therefore,  also  in  anti-pneu- 
mococcic  serum,  and  that  unless  controls  be  made  false  conclusions 
might  be  drawn  from  this.  In  their  homologous  sera  they  may 
agglutinate  in  high  dilutions.  Another  point  made  clear  is  that 
various  streptococci  of  the  same  morphological  and  possibly 
even  the  same  fermentative  and  cultural  characters  do  not,  with 
anything  like  the  uniformity  displayed  by  pneumococcus  cul- 
tures, agglutinate  in  a  given  streptococcus  serum.  This  indicates 
the  possibility  of  a  future  satisfactory  classification  on  this  basis. 

The  other  organisms  of  the  series,  which  have  not  as  yet  been 
mentioned  but  which  deserve  especial  attention,  are  inulin- 
fermenters  of  pneumococcus  morphology  but  without  capsules, 
other  lancet  organisms  not  so  definitely  of  the  pneumococcus 


16-i         Comparative  Study  of  Pneumococci  and  Allied  Organisms 

type,  and  organisms  apparently  of  definite  streptococcus  mor- 
phology, all  of  which  ferment  inulin. . 

Some  of  the  first  variety,  if  not  all,  are  undoubtedly  pneumo- 
cocci, and  agglutinate  in  pneumococcus  immune  serum,  though 
usually  not  in  high  dilutions,  and  there  seems  no  reason  for 
placing  them  in  a  separate  class. 

Other  inulin  fermenters,  which  are  small  organisms,  at  times 
showing  lancet  morphology,  but  which  are  usually  non-capsu- 
lated,  and  vary  in  some  of  the  less  important  features  in  artificial 
media,  it  is  difficult  to  classify,  as  they  show  little  or  no  agglu- 
tination in  pneumococcus  serum.  Whether  they  are  modified 
pneumococci  or  should  be  placed  in  a  distinct  class,  our  studies 
at  the  present  writing  have  failed  to  determine.  Their  agglu- 
tinations are  recorded  in  the  table.  Apparently  all  of  them  are 
not  of  the  same  type.  If  the  requisite  time  had  been  at  our  dis-  . 
posal,  a  special  study  of  the  pathogenic  effects  of  these  organisms 
on  the  one  hand,  and  on  the  other  of  the  modifying  influence  on 
the  organisms  themselves  of  a  residence  in  the  animal  body, 
would  have  been  undertaken. 

Our  attention  has,  however,  been  especially  directed  to  certain 
non-capsulated,  inulin-fermenting  cultures  kindly  sent  to  us  by 
Dr.  Charles  Norris  and  Dr.  Leo  Buerger.  Two  of  the  cultures 
from  Dr.  Norris  show  variations  from  the  classic  pneumococcus 
type.  One  of  these,  known  by  us  as  "Nor.  199,"  only  varies 
from  this  type  in  not  possessing  a  capsule.  Its  morphology 
otherwise  is  typical,  and  its  growth  in  rabbit  blood-agar  c\il- 
tivations  corresponds  absolutely  to  the  growth  given  by  the  vast 
majority  of  pneumococci  in  this  medium.  It  agglutinates  to 
some  extent  in  pneumococcus  immune  serum  and  is  probably  a 
true  pneumococcus. 

The  other  culture,  "  Nor.  102,"  has  no  capsules  and  shows  a  less 
typical  lancet  morphology.  Its  growth  in  blood-agar  plates  is 
not  that  of  the  most  diagnostic  type,  but  corresponds  to  a  type 
seen,  nevertheless,  among  certain  otherwise  perfectly  typical 
pneumococci.  No  agglutination  in  pneumococcus  serum  has 
been  shown.  The  weight  of  evidence  is,  however,  in  favor  of  its 
being  a  temporarily  or  permanently  modified  pneumococcus. 


Philip  Hanson  Hiss  165 

Dr.  Buerger's  culture,  laiown  as  "Streptococcus  No.  7,"  fer- 
ments inulin.  Its  morpholog}^  is  in  general  of  streptococcus 
type,  but  lancet  forms  at  times  may  predominate.  Very  narrow 
capsules  have  been  observed  by  me  on  some  cultivations  of  this 
organism.  This  organism  does  not  agglutinate  to  an  appre- 
ciable degree,  according  to  our  macroscopic  tube  agglutinations, 
in  anti-pneumococcus  sera,  but  gives  high  agglutination  in  its 
homologous  serum.  Some  cultures  of  pneumococci  agglutinate 
in  this  serum  even  in  high  dilutions.  In  blood-agar  plates  the 
colonies  appear  brown  or  dark-green  surrounded  by  an  opaque 
area — the  whole  plate  assuming  a  distinct  greenish  tinge,  which 
is  more  marked,  if  anything,  than  the  tinge  occurring  in  most 
pneumococcus  plates. 

The  organism  is  definitely  not  a  streptococcus  of  the  pyogenes 
type,  and  probably  not  a  true  streptococcus,  unless  it  is  of  the 
type  described  by  SchottmuUer  as  Streptococcus  mitior  viridans. 
Not  having  had  the  opportunity  of  examining  Schottmiiller's 
original  cultures,  it  is,  of  course,  not  known  to  us  whether  they 
ferment  inulin  or  are  non-inulin  fermenters  of  streptococcus 
type,  such  as  have  been  met  with  in  our  present  studies,  and 
which  also  produce  a  greenish  color  but  no  clear  lysis  in  blood- 
agar  plates.  The  true  status  of  this  Buerger  streptococcus  (?) 
we  have,  therefore,  up  to  the  present  been  unable  satisfactorily 
to  determine,  though  it  may  be  an  atypical  pneumococcus. 

GROWTH    CHARACTERS    IN    BLOOD-AGAR. 

SchottmuUer  (3)  in  1903  and,  independently  of  him,  Rosenow 
(4)  in  1904  called  attention  to  certain  reactions  caused  by  pneu- 
mococci and  streptococci  when  growing  in  agar  with  which 
human  or  animal  blood  had  been  mixed. 

SchottmuUer  stated  that  pneumococcus  colonies  developing  in 
this  medium  usually  became  of  a  greenish  tinge,  and  were  sur- 
rounded by  a  zone  of  opacity  of  a  greenish  color.  Strepto- 
cocci of  the  erysipelatos  type  did  not  asstune  a  dark  or  greenish 
tinge,  and  were  surrounded  by  a  distinct  clear  zone  due  to  com- 
plete lysis  of  the  red  corpuscles  and  change  of  the  hemoglobin. 
Rosenow 's  observations  confirmed  these. 


166         Com'parative  Study  of  Pneu')nococci  and  Allied  Organisms 

SchottniuUer  further  described  a  form  of  streptococcus  which 
he  designated  Streptococcus  mitior  viridans.  This  was  less 
virulent  than  the  other  type  and  was  said  to  be  usually  asso- 
ciated with  rather  chronic  lesions  and  septiccemias.  It  was  de- 
scribed as  of  streptococcus  morphology  and  non-capsulated. 
In  blood-agar  plates  it  simulated  the  pneumococcus,  but  the 
greenish  tinge  was  less  intense.  Little  or  no  haemolysis  occurred. 
What  the  true  nature  of  these  organisms  is,  is  in  doubt,  as  nothing 
is  known  of  their  fermentations  and  agglutinations. 

In  our  series  of  organisms  we  have  found  the  action  of  pneu- 
mococci,  as  a  rule,  to  conform  to  Schottmiiller's  description,  but 
usually  with  a  deep-brown  or  reddish  tinge  to  the  colonies  rather 
than  a  definite  green,  the  opaque  area  of  partial  hemolysis 
(and  precipitation  (?)  ),  but  not  clearing,  and  of  yellowish  or 
greenish  tinge  being  present. 

Some  organisms  of  our  series  of  true  non-inulin-fermenting 
streptococcus  type  conformed  to  his  description  of  Streptococcus 
erysipelatos  with  the  clear  zone  surrounding  their  colonies  and 
no  greenish  tinge.  Other  non-inulin-fermenting  organisms  of 
general  streptococcus  type,  some  capsulated  (see  P.  and  S., 
No.  8)  and  some  not  capsulated,  gave  the  typical  pneumococcus 
or  Streptococcus  mitior  pictures,  which  are  really  not  to  be 
distinguished  from  each  other. 

Our  unclassified  inulin-fermenting  organisms  either  produced 
no  lysis  or  change  in  color  of  the  medium,  or  gave  rise  to  appear- 
ances closely  suggesting  or  absolutely  corresponding  to  those 
given  by  pneumococci. 

Whether  this  can  be  depended  upon  as  indicating  their  pneu- 
mococcus nature,  one  would  hesitate  to  say  in  face  of  the  fact  that 
so  many  non-inulin-fermenting,  non-agglutinating,  and  definitely 
not  pneumococcus  cultures  also  give  rise  to  these  appearances. 

In  streak  cultures  on  slanted  rabbit-blood-agar  in  tubes, ^  the 
writer,  in  making  a  comparative  observation  on  about  two  hun- 

'  This  medium,  is  made  by  bleeding  a  rabbit  from  the  carotid  artery,  through 
a  sterile  canula  and  rubber  tube,  directly  into  tubes  of  fluid  sterile  agar  (kept 
at  from  45°  to  50°  C.)-  About  one-half  to  one  cubic  centimeter  of  blood  should 
be  added  to  each  tube.  The  blood  must  be  mixed  immediately  with  the  agar 
(to  prevent  clotting),  and  the  tubes  slanted  and  allowed  to  harden. 


Philip  Hanson  Hiss  167 

dred  pneumococci,  streptococci,  and  miscellaneous  unidentified 
organisms,  noted  certain  appearances  which,  when  prominent, 
seemed  to  be  practically  diagnostic  of  the  pneumococcus  cul- 
tures. 

Streak  cultures  of  pneumococci  on  mixed  blood-agar  grow  at 
first  with  the  usual  characters  seen  on  serum  media.  The  growth, 
however,  appears  usually  of  a  rather  dirty  yellowish  tinge,  and 
the  blood-agar  in  immediate  contact  with  the  growth  and  at 
times  for  some  distance  from  it  takes  on  a  yellowish  rather 
opaque  look,  due  to  decolorization  and  probably  to  a  slight  pre- 
cipitation from  acid  formation.  The  variations  met  with  are 
usually  in  the  direction  of  a  less  typical  growth  layer,  combined 
with  more  decolorization  and  haemolysis,  though  the  medium 
remains  opaque. 

During  the  first  few  days  of  growth  neither  of  these  appear- 
ances is  characteristic  When,  however,  the  tubes  have  remained 
in  the  incubator  some  days,  the  majority  of  the  pneumococcus 
cultures  take  on  a  distinctly  characteristic  appearance.  Most 
of  the  growth  disappears,  leaving  the  general  decolorized  dirty- 
yellowish  area,  while  definite  raised  colony -like  nodular  masses 
remain,  which  usually  are  of  a  brownish-black  or  dark-red  color, 
as  if  the  masses  had  become  distinctly  stained  with  the  blood 
pigments.  One  gets  the  impression  of  a  blistered  painted  sur- 
face. None  of  the  streptococci  or  other  organisms,  so  far  as 
determined  by  this  test,  assumed  this  appearance.  Whenever 
this  appearance  was  noted,  the  organisms  were  found  to  be  true 
pneumococci.  The  cultures  of  pneumococci,  giving  less  typical 
growth  and  more  extensive  decolorization  of  the  medium,  re- 
tained the  more  even  appearance  of  surface  and  did  not  become 
nodular.  So  far  as  we  know,  these  cultures  are  true  pneumo- 
cocci, but,  it  may  possibly  be  that  they  are  modified  Streptococcus 
mucosus  cultures  which  had  not  previously  been  recognized. 
The  Streptococcus  mucosus  growth,  though  at  first  prominent 
and  moist  and  quite  characteristic  on  this  medium,  soon  prac- 
tically disappears.  Traces  of  decolorization  may  or  may  not  be 
prominent.  One  culture,  however,  presented  an  appearance 
similar  to  that  of  the  pneumococcus. 


168         Comparative  Study  of  Pneumococci  and  Allied  Organisms 

PATHOGENICITY. 

This  phase  of  study  has  not,  as  was  stated  earlier,  been 
taken  up  with  any  regularity  in  our  comparative  study. 
Simple  tests  of  the  pathogenicity  of  organisms,  especially  such 
organisms  as  pneumococci  and  streptococci,  usually  give  little 
information  of  value  in  classification,  and  even  definite  studies 
of  lesions  caused  by  such  organisms  are  not  often  of  diagnostic 
aid. 

It  has  been  generally  demonstrated  by  all  investigators  that 
pneumococci  as  a  class,  especially  when  freshly  isolated,  are 
usually  pathogenic,  although  in  varying  degrees,  for  white  mice 
and  rabbits ;  while  streptococci,  even  from  severe  lesions  in  man, 
may  show  little  or  no  pathogenicity  for  mice  and  rabbits  and 
other  test  animals.  Both  of  these  organisms,  even  when  pri- 
marily virulent  in  high  degree,  tend  to  lose  this  character  when 
cultivated  on  the  usual  artificial  media.  Any  tests,  therefore, 
to  determine  the  pathogenicity  of  the  organisms,  when  they 
arrived  at  our  laboratory,  would  have  led  to  little  or  no  infor- 
mation of  value  as  to  the  original  pathogenicity  of  the  cultures. 
This  work  was  left  in  the  hands  of  the  investigators  who  made 
the  isolations. 

All  that  need  be  said  here  is  that  so  far  as  our  own  cultures  are 
concerned  and  so  far  as  we  can  gather  from  the  information 
sent  to  us  by  the  workers  in  other  places,  little  or  no  difference 
has  been  determined  in  the  relative  pathogenicity  of  organ- 
isms of  pneumococcic  type  from  the  mouths  of  healthy  indi- 
viduals and  those  from  persons  suffering  from  pneumococcic 
infections. 

If  such  pathogenicity  for  animals  indicates  in  any  way  the 
grade  of  pathogenicity  of  organisms  for  man, — a  supposition 
always  open  to  grave  doubt, — and  if,  especially,  these  organisms 
be  found  to  linger  in  normal  mouths  during  the  summer  months, 
the  possession  of  virulency  by  organisms  from  healthy  individuals 
is  a  matter  of  interest  and  importance  in  the  consideration  of  the 
mouth  as  a  nidus  of  these  infective  agents  and  the  possible  and 
probable  ways  of  their  preservation  and  dissemination. 


Philip  Hanson  Hiss  169 

REMARKS     ON    THE     CLASSIFICATION     OF    THE     ORGANISMS     INVES- 
TIGATED. 

An  attempt,  at  this  stage  of  our  work,  to  classify  definitely  all 
the  organisms  which  have  been  received  by  us  would  be  ill- 
advised.  Many  of  these  have  but  recently  come  into  our  hands, 
and  when  not  responding  definitely  to  the  tests  and  showing 
easily  recognized  and  diagnostic  characters  of  some  one  of  the 
well-known  types,  our  acquaintance  with  them  has  been  too 
limited  to  warrant  an  attempt  at  classification.  Any  points, 
however,  so  far  determined  about  their  general  morphology  and 
biology,  are  recorded  in  the  table. 

The  following  tentative  classification  is  given  for  purposes  of 
reference  and  as  an  illustration  of  some  of  the  types  met  with : 

A .     Inulin  fermenters. 

1.  Typical  pneumococcus  morphology.  Typical  capsules. 
Agglutinate  in  pneumococcus  immune  serum.  Growth 
on  or  in  blood-agar  usually  typical.     Typical  pneumococci. 

2.  Typical  pneumococcus  morphology.  No  capsules  de- 
termined, even  in  body  fluids  of  infected  animals.  Ag- 
glutinate in  pneumococcus  immune  serum  but  usually  not 
in  very  high  dilutions.  Growth  in  blood-agar  same  as 
that  of  typical  pneumococci.     Pneumococci. 

3.  Atypical  morphology.  Lancet-shaped  organisms  occur, 
but  morphology  approximates  streptococcus  type,  or 
some  cultures  may  be  composed  of  small  lancet-shaped 
diplococci.  Capsules,  usually  not  well-marked,  may  be 
present.  Growth  in  blood-agar  plates  may  or  may  not 
be  typical.  Do  not  show  nodular  growth  on  blood-agar 
streak  ctiltivations.  Organisms  mostly  from  pneumonic 
lungs  at  autopsy  or  from  some  internal  body  source.  Do 
not  ordinarily  show  diagnostic  agglutinations  in  pneumo- 
coccus immune  serum,  but  may  agglutinate  in  homolo- 
gous sera  to  fair  degree.  Pneumococci  do  not,  as  a  rule, 
agglutinate  diagnostically  in  these  sera.  Probably  a 
mixed  group,  some  of  which  are  presumably  true  but 
atypical  pneumococci,  or  atypical  Streptococcus  mucosus. 

4.  Morphology  practically  like  that  of  the  pneumococcus. 


170          Comjjarative  Study  of  Pneumococci  and  Allied  Organisms 

but  chains  are  more  frequent  and  the  elements  are  more 
usually  spherical.  Capsules  typical  and  well-marked. 
Agglutinates  only  in  low  dilutions  of  homologous  immune 
serum  or  in  pneumococcus  immune  serum.  Pnetimo- 
cocci  agglutinate  in  high  dilutions  of  the  serum  of  animals 
inoculated  with  this  type.  Separated  from  pneumococci 
by  peculiarities  of  growth  in  gelatin  and  on  Loeffler's  and 
other  seram  media.  Typical  Streptococcus  mucosus:  prob- 
ably a  variety  of  pneumococcus. 

B.     Non-inuUn  fermenters. 

5.  Capsulated  organisms  simulating  pneumococci,  but  of 
general  streptococcus  morphology.  Capstdes  fairly  con- 
stant on  artificial  media.  Ferment  many  sugars,  but  not 
inulin.  Simulate  pneumococcus  growth  and  reactions  in 
blood-agar  plates,  but  do  not  show  nodular  growth  on 
blood-agar  streak  cultivations.  Do  not  agglutinate  in 
pneumococcus  immune  serum.  Agglutinate  in  moderate 
dilutions  of  homologous  serum,  in  which  serum  the  pneu- 
mococcus does  not  agglutinate.  Streptococcus  mitior 
viridans  (?). 

6.  True  streptococcus  morphology.  Ordinarily  capsules  are 
not  demonstrable.  Ferment  many  sugars  but  never 
inulin.  Colonies  in  blood-agar  plates  not  green-tinged, 
but  surrounded  usually  by  a  well-marked  clear  zone.  Apt 
to  agglutinate  in  normal  rabbit  serum,  even  up  to  i :  200. 
Agglutinate  markedly  in  homologous  sera.  Pneumo- 
cocci are  practically  not  agglutinated  by  these  sera. 
Typical  Streptococcus  pyogenes  {erysipelatos) . 

7.  Streptococcus  morphology.  May  or  may  not  have  cap- 
sule. Ferment  many  sugars.  Orange  growth  along 
puncture  in  Welch's  and  other  media.  Agglutinations 
in  general  not  determined,  but  do  not  agglutinate  in 
pneumococcus  serum.     May  be  mixed  group. 

8.  Streptococcus  morphology.  May  or  may  not  have  cap- 
sules. Sugars  not  fermented,  with  possible  exception  of 
some  monosaccharids.  Do  not  agglutinate  in  pneumo- 
coccus immune  serum. 


Philip  Hanson  Hiss  171 

It  should  be  noted  in  connection  with  this  classification  of  the 
streptococci,  that  it  is  merely  in  outline,  and  that  other  classifi- 
cations, possibly  of  value,  are  to  be  arrived  at  from  a  detailed 
grouping  by  fermentation  reactions.  Attention  was  called  to 
this  subject  by  the  writer  in  a  previous  paper.  A  close  study  of 
agglutination,  taken  in  connection  with  the  groupings  suggested 
by  fermentation  reactions,  will  probably  lead  to  a  separation  of 
streptococci  into  distinct  and  easily  recognizable  groups. 

So  far  as  the  whole  classification  is  concerned,  it  should  again 
be  recalled  that  variations  of  a  fairly  permanent  or  even  of  a 
transient  nature  in  the  morphology  and  physiology  of  organisms 
may  definitely  interfere  with  their  recognition,  and  when  classi- 
fications have  to  be  based  on  such  delicate  biological  processes 
as  are  involved  in  fermentations  and  agglutinations,  only  long- 
extended  and  painstaking  observations  will  lead  to  an  eventual 
recognition  of  their  true  identity. 

In  connection  with  this  statement  and  the  following  remarks 
upon  statistics,  it  is  of  value  to  note  the  percentage  of  discrep- 
ancies between  the  results  obtained  by  us  from  our  examination 
of  the  cultures  sent  to  the  central  laboratory  and  the  results 
which  were  obtained  by  the  various  workers  from  their  examina- 
tion of  these  same  cultures  and  reported  to  us.  Out  of  146  in- 
stances in  which  the  cultures  were  reported  as  fermenting  inulin, 
we  have  only  been  able  to  determine  the  occurrence  of  such 
fermentation  in  the  case  of  119  of  the  cultures,  a  difference  of 
i8|-  per  cent.  The  results  of  our  inulin  determinations  were, 
nearly  without  exception,  supported  by  the  agglutination  tests 
— in  only  two  instances,  in  fact,  where  inulin  fermentation  was 
reported  and  our  tests  gave  negative  results  did  the  agglutination 
results  seem  to  indicate  the  pneumococcus  nature  of  the  organ- 
isms. In  both  of  these  cultures  the  organisms  had  a  distinct 
streptococcus  morphology,  and  as  streptococci  frequently  ag- 
glutinate in  high  dilutions  of  normal  and  heterologous  sera  these 
results  are  not  determinative. 

In  only  one  instance  did  we  find  inulin  fermentation  when 
none  was  reported,  and  in  this  culture  the  morphology  was 
perfectly    typical,    and    typical    capsules    were    present.      Our 


172         Comparative  Study  of  Pneumococci  and  Allied  Organisms 

results  show  in  this  instance,  however,  that  no  agglutination 
took  place  in  pneumococcus  immune  serum. 

This  discrepancy  of  nearly  twenty  per  cent.,  indicating  as  it 
does  the  differences  in  identifications  of  the  same  organisms 
when  studied  by  different  investigators,  has  an  extremely  im- 
portant bearing  upon  the  percentage  of  error  which  in  the 
ordinary  course  of  events  must  be  allowed  in  the  consideration 
of  statistics  from  various  sources, 

STATISTICAL. 

Bacteria  of  Pneumococcus  Type  in  Normal  Mouths. — Enough 
has  been  said  in  the  previous  part  of  this  paper  to  make  it  clear 
that  there  occur  in  the  mouths  of  healthy  persons  organisms  that 
show,  according  to  the  most  refined  morphological  and  biological 
tests,  all  the  characters  displayed  by  true  pneumococci  from 
pathological  sources.  No  reasonable  doubt  can  now  exist  as  to 
their  true  pneumococcus  character. 

One  of  the  fundamental  objects  of  the  bacteriological  work 
instituted  by  the  Commission — the  solution  of  the  problem  of 
the  nature  of  these  organisms — may  therefore  be  looked  upon  as 
accomplished. 

Allied  to  this  a  point  of  great  importance  has  also,  we  think, 
been  conclusively  demonstrated,  namely,  other  organisms  are  to 
be  met  with  which  too  closely  simulate  pneumococci  morpho- 
logically to  be  separated  from  them  by  the  most  careful  mor- 
phological examination  or  by  the  usual  routine  cultural  tests, 
only  fermentation  and  agglutination  tests  definitely  establishing 
their  nature.  The  futihty  of  attempting  to  base  statistics  on 
purely  morphological  findings  and  staining  reactions,  or  even 
superficial  cidture  tests,  must,  therefore,  be  obvious.  At  the 
best  they  are  but  indicative.  ^'^ 

Another  object  of  the  Commission,  necessarily  secondary  to 
the  establishing  of  the  nature  of  mouth  organisms,  was  the  de- 

'  0  Old  statistical  statements  as  to  the  frequency  of  occurrence  of  pneumococci 
in  normal  mouths,  although  this  frequency  may  be  found  to  approximate 
closely  present  findings,  must,  nevertheless,  in  the  light  of  these  facts,  lose  any 
just  claim  to  scientific  accuracy. 


Philip  Hanson  Hiss  173 

termination  of  the  frequency  of  occurrence  of  pneumococci  in 
the  mouths  of  healthy  individuals. 

For  such  studies  to  be  of  definite  statistical  value  it  was  ob- 
vious that  series  of  individuals  living  under  different  environ- 
mental conditions  should  be  examined.  These  were  indicated 
as  follows : 

1.  Normal  persons,  giving  history  of  no  known  contact  with  a 
source  of  infection  and  no  recent  history  of  "cold,"  bronchitis, 
or  pneumonia,  middle  ear,  conjunctival,  or  other  possible  pneu- 
mococcic  infection. 

2.  Normal  persons,  intimately  associated  with  patients  suffer- 
ing from  pneumococcic  infections — i.e.,  pneumonia,  bronchitis, 
etc.  (nurses,  doctors,  hospital  attendants,  and  non-pneumonic 
patients  in  wards  with  pneumonia  patients) . 

These  investigations  were  not  to  be  confined  to  one  locality, 
but  to  be  carried  on  in  other  places  besides  New  York — in  par- 
ticular in  Philadelphia,  Boston,  and  Chicago, — thus  assuming  a 
much  more  general  significance,  and  in  themselves  affording  a 
basis  of  comparison  of  frequency  of  occurrence  in  different 
localities.  The  results  of  this  investigation  by  the  various 
workers  will  doubtless  eventually  be  correlated  and  tabulated ;  at 
present  they  are  scattered  throughout  the  independent  reports. 

Before  considering  the  results  of  our  own  investigations  along 
these  lines,  attention  should  be  called  to  certain  factors  that 
may  influence  deductions  made  from  such  findings.  Con- 
sidered from  the  standpoint  of  a  simple  illustration  of  the  ease 
of  general  dissemination  of  organisms  thrown  off  from  the 
bodies  of  diseased  persons  by  way  of  the  mouth  and  nose,  the 
finding  of  pneumococci  in  the  mouths  of  normal  individuals  may 
seem  to  have  great  significance.  Another  interpretation  is, 
however,  perfectly  legitimate  from  such  simple  data;  in  other 
words,  these  organisms  may  be  common  and  permanent  in- 
habitants of  mouths,  as  colon  bacilli  are  supposed  to  be  of  the 
digestive  tract.  If  such  a  view  were  substantiated,  the  appli- 
cation of  protective  hygienic  measures  might  assume  less  im- 
portance. 

There  seem  to  be  two  definite  ways  of  solving  this  problem. 


174        Comparative  Study  of  Pneumococci  and  Allied  Organisms 

Of  these  the  only  one  open  to  the  investigator  in  cities  is  the 
careful  examination  over  extended  periods  of  the  bacterial  flora 
of  the  mouth  of  one  and  the  same  individual.  In  this  way  the 
permanent  flora  of  such  a  mouth  may  soon  be  determined  and 
the  appearance  or  disappearance  of  organisms  noted  with  accu- 
racy. Even  varieties  of  organisms  of  the  same  species  may 
be  recognized,  when  thoroughly  investigated,  by  some  minor 
biologiqal  or  possibly  morphological  peculiarity,  and  thus  defi- 
nitely distinguished  from  a  new  invader  of  the  same  species.  If, 
then,  upon  the  advent  of  such  an  invader  pathologic  changes 
occurred,  its  association  with  these  would  be  fairly  well  estab- 
lished, and  the  entire  period  of  its  residence  could  be  determined. 
The  other  method  of  solving  this  problem  is  the  investigation 
of  individuals  living  in  localities  where  pneumonia  does  not 
ordinarily  occur,  or  of  individuals  not  coming  frequently  into 
contact  with  large  masses  of  population.  As  a  possible  means 
of  arriving  at  a  solution  of  this  problem,  the  writer  has  sug- 
gested the  examination  of  the  mouths  of  such  persons  as  sailors 
arriving  from  long  voyages  by  sea.^^ 

By  such  methods  as  these  the  permanency  of  the  occupation  of 
the  normal  mouth  by  such  organisms  could  be  determined,  or 
their  definitely  transient  nature  be  shown.  This  will  be  referred 
to  again  in  considering  the  statistics  of  our  own  investigations. 

Our  own  investigations  on  the  bacterial  flora  of  the  mouths  of 
normal  persons  was  limited  to  a  study  of  the  saliva  from  twenty- 
two  persons  not  known  to  be  suffering  from  any  form  of  pneu- 
mococcic  infection.  In  the  case  of  fifteen  of  these  individuals 
only  one  examination  was  made,  and  this  in  each  instance  by 
mouse  inoculation.  If  the  mouse  did  not  die  within  a  reasonable 
time  the  bacteria  of  the  local  lesions  were  investigated.  Organ- 
isms of  definite  pneumococcus  type  were  isolated  from  seven  of 
the  fifteen  persons,  i.e.,  in  46.6  %,  The  saliva  from  the  remain- 
ing seven  persons  was  tested  repeatedly,  and  pneumococci  were 
demonstrated  in  the  saliva  of  six  out  of  the  seven,  i.e.,  in  85.7%. 

'  >  This  work  is  now  being  carried  on  at  the  New  York  Quarantine  Station, 
and,  it  is  hoped,  may  throw  some  Hght  upon  the  subject  of  the  persistence  of 
pneumococci  in  normal  mouths. 


Philip  Hanson  Hiss  175 

These  higher  figures  may  be  interpreted  in  two  ways,  either  as 
representing  a  correction  of  errors  due  to  the  single  application 
of  any  given  method  of  isolating  organisms,  or  to  this  in  part 
and  in  part  to  the  detection  of  new  invaders,  since  the  examina- 
tions were  made  at  intervals,  over  several  months.  No  matter 
what  the  actual  interpretation,  the  prominent  fact  brought  out 
is  that  practically  every  individual,  at  least  during  the  winter 
season  when  exposed  to  environmental  conditions  such  as  those 
existing  in  New  York  City,  acts  as  host,  at  some  time  or  other, 
and  probably  at  repeated  intervals,  for  organisms  of  the  most 
characteristic  pneumococcus  type.  In  answer  to  the  question 
above  proposed  as  to  the  continuous  residence  of  such  organisms 
as  parasites  of  the  mouths  of  normal  persons,  it  may  be  said  that 
it  is  the  writer's  opinion  that  in  the  majority  of  cases  the  resi- 
dence of  any  given  strain  of  pneumococci  under  such  conditions 
is  apt  to  be  temporary  rather  than  permanent  or  long  continued. 

Some  of  the  reasons  for  such  an  opinion  may  be  given :  in  the 
first  place,  repeated  examinations  carried  on  by  all  methods  at 
short  intervals  may  fail  for  a  longer  or  shorter  time  to  demon- 
strate the  presence  of  pneumococci  in  the  mouths  of  a  certain 
percentage  of  normal  individuals.  This,  taken  in  connection 
with  their  practically  universal  occiurence,  indicates  that  or- 
ganisms of  this  type  tend  to  disappear  from  such  mouths ;  and 
furthermore,  even  when  pneumococci  are  found  repeatedly  and 
constantly  throughout  long  periods,  unless  the  strain  present  has 
some  minor  peculiarity  by  which  it  may  be  recognized  from  new 
invaders,  we  cannot  argue  that  we  are  not  dealing  with  newly 
acquired  organisms  at  each  examination. 

In  the  second  place,  unless  such  sensitive  organisms  rapidly 
become  adapted  to  some  more  or  less  protected  nidus  in  the 
mouth  or  naso-pharynx,  such  as  the  tonsillar  crypts  or,  as  it 
seems  not  unlikely  to  the  writer,  to  a  parasitic  residence  in  the 
salivary  ducts,  the  ordinary  conditions  in  the  mouth  during  the 
taking  of  foods,  especially  of  an  acid  nature,  and  the  rapid 
osmotic  changes  to  which  the  organisms  in  general  must  be 
subjected  when  food  or  drink  is  in  the  mouth,  seem  highly 
unfavorable  for  organisms  so  sensitive  to  their  environment  as 


176        Comparative  Study  of  Pneumococci  and  Allied  Organisms 

pnetunococci  undoubtedly  are  under  observed  cultural  condi- 
tions. Further  than  this,  flora  already  adapted  may  interfere 
with  the  occupation  by  these  new  forms.  It  seems  not  un- 
likely, therefore,  that  such  invaders  usually,  unless  particularly 
favorable  conditions  (temporary  or  permanent)  for  their  adapta- 
tion to  this  new  environment  exist,  would  tend  to  die  off  after  a 
comparatively  short  residence. 

PNEUMOCOCCI    IN   THEIR    RELATION    TO    "  COLDS." 

When  the  repeated  investigation  of  the  flora  of  the  same 
mouth  was  taken  up,  we  had  in  view  not  only  the  determination 
of  the  normal  and  transient  bacterial  inhabitants,  but  also  of 
the  relation  of  these  latter  to  disease  processes  which  might 
manifest  themselves  during  the  period  of  such  examinations.  In 
most  instances,  so  far  as  the  histories  showed,  no  "  cold,"  pharyn- 
gitis, bronchitis,  or  pneumonia,  either  preceded,  within  a  recent 
period,  the  determination  of  pneumococci  in  the  mouth,  or 
developed  subsequent  to  the  invasion  of  the  mouth  or  naso- 
pharynx by  these  organisms.  In  two  instances,  however,  the 
appearance  and  residence  of  pneumococci  in  the  mouths  of 
persons  hitherto  free  from  them  were  incident  with  the  develop- 
ment and  course  of  "colds."  Subsequent  examinations  in  one 
case  showed  the  early  disappearance  of  the  organisms ;  in  the 
other,  in  which  the  inflammatory  process  had  been  much  more 
severe  and  a  purulent  post-nasal  discharge  was  developed,  the 
organism  (presumably  the  same)  had  remained  in  the  mouth  up 
to  the  last  examination,  about  a  month  subsequent  to  the  cessa- 
tion of  all  symptoms  of  infection.  In  the  purulent  discharge 
pneumococci  were  present  in  practically  pure  culture  and  in  the 
greatest  profusion. 

In  each  of  these  cases,  it  is  interesting  to  note  as  an  illustration 
of  the  value  of  careful  study  in  recognizing  organisms,  that  the 
mouth  seemed  to  be  the  permanent  habitat  of  organisms  of  general 
pneumococcus  morphology — in  one  instance  of  a  Gram-positive, 
capsulated,  non-inulin-fermenting  streptococcus  morphologically 
not  definitely  to  be  distinguished  from  the  pneumococcus;  in 
the  other,  of  a  typical  Streptococcus  mucosus.     In  both  cases 


Philip  Hanson  Hiss  177 

these  organisms  were  found  upon  the  first  examination,  and  per- 
sisted throughout  the  entire  period  of  examination  extending 
over  months.  The  capsulated  streptococcus  rarely  killed  mice, 
but  could  always  be  isolated  from  the  local  lesion.  The  Strepto- 
coccus mucosus,  on  the  other  hand,  was  pathogenic,  and  found 
in  the  blood  of  the  infected  mouse;  when,  however,  the  pneu- 
mococcus  invaded  the  mouth,  the  inoculated  mice  died  of  a  pure 
pneumococcus  septicaemia  (so  far  as  could  be  determined  from 
smears  on  serum  plates) ,  while  the  Streptococcus  mucosus  could 
be  recovered  from  the  local  lesion  or  from  direct  smears  of 
saliva  on  serum-agar  plates. 

One  other  series  of  examinations  is  of  interest  in  connection 
with  the  persistence  of  certain  types  of  organisms  in  the  mouth. 
In  this  case  the  mice  throughout  the  entire  period  of  examination 
invariably  died  from  a  septicemia  caused  by  a  Gram-negative, 
capsulated  bacillus  of  the  Friedlander  type.  The  first  saliva 
was  tested  December  i,  1904,  the  last  on  April  28,  1905, — a 
period  of  six  months.  No  pneumococcus  was  ever  isolated  from 
this  person,  but  in  smear  plates  from  the  saliva  on  serum-agar  a 
Gram-positive,  capsulated  coccus,  displaying  the  characteristic 
growth  appearance  of  Streptococcus  mucosus,  was  observed. 

These  observations  suggested  to  the  writer  the  probability, 
mentioned  before,  of  such  organisms  being  permanent  residents 
of  the  protected  salivary  ducts  or  the  tonsillar  crypts,  or  possibly 
being  accounted  for  by  their  connection  with  slight  suppurative 
processes  of  the  teeth  and  gums. 

SUMMARY    AND    CONCLUSIONS. 

The  work  carried  on  under  the  auspices  of  the  Medical  Com- 
mission for  the  Investigation  of  Acute  Respiratory  Diseases,  at 
the  Bacteriological  Laboratory  of  the  College  of  Physicians  and 
Surgeons,  Columbia  University,  has  consisted  principally  of  a 
comparative  study  of  the  morphology,  growth  characters,  fer- 
mentative activities,  and  agglutination  reactions  of  pneumo- 
cocci  and  allied  organisms  isolated  by  ourselves  and  the  various 
workers  under  the  Commission. 

These  organisms  were  from  two  chief  sources :  (a)  from  the 


178        Comparative  Study  of  Pneumococci  and  Allied  Organisms 

mouth  and  naso-pharynx  of  supposedly  normal  persons  and  per- 
sons suffering  from  minor  inflammations  of  the  naso-pharynx,  and 
(&)  from  definitely  pathologic  sources,  such  as  pneumonic  sputum, 
pneumonic  lungs,  empysema,  the  circulating  blood  of  pneujnonia 
patients,  septicaemias,  meningitis,  and  various  minor  lesions, 
usually  due  to  infection  with  pneumococci. 

One  of  the  principal  objects  of  the  study  was  to  make  a  care- 
ful comparison,  in  the  light  of  the  most  recent  knowledge  of  the 
biology  of  the  pneumococcus  and  by  the  aid  of  the  latest  biologi- 
cal and  technical  methods,  of  the  series  of  organisms  from  these 
two  sources,  and  thus  to  determine  definitely  the  true  nature  of 
pneumococcus-like  organisms  occurring  in  the  mouths  of  normal 
persons. 

The  second  and  equally  important  object,  ultimately  dependent, 
however,  upon  the  solution  of  the  first,  was  the  determination 
of  the  frequency  of  occurrence  of  typical  pneumococci  in  the 
mouths  of  healthy  individuals. 

The  investigations  detailed  in  the  present  paper  have  dealt 
chiefly  with  the  first  problem  and  incidentally  with  the  second, 
and  lead  to  the  following  conclusions : 

(a)  That  organisms,  not  to  be  distinguished  by  morphological 
characters  or  by  any  physiological  peculiarities  from  true  pneu- 
mococci derived  from  pathologic  sources,  occur  with  frequency 
in  the  mouths  of  healthy  persons  and  those  suffering  from  slight  in- 
flammations of  the  naso  pharynx,  and  that  the  only  permissible  and 
legitimate  conclusion  is  that  these  organisms  are  true  pneumococci, 

(6)  That  there  are  other  organisms  in  normal  mouths  and 
from  pathologic  sources  that  morphologically  or  by  staining 
reactions  are  not  definitely  to  be  distinguished  from  pneumo- 
cocci, and  can  only  be  recognized  by  a  careful  study  of  their 
fermentative  activities  and  agglutination  reactions.  These 
organisms  are  non-inuHn  fermenters. 

{c)  That  the  organism  known  as  Streptococcus  mucosus  is 
at  times  found  in  cultivations  from  the  mouths  of  apparently 
healthy  individuals,  and  that,  although  it  shows  certain  pecuH- 
arities  distinguishing  it  from  the  typical  pneumococcus,  it  is 
probably  very  closely  related  to,  and  a  variety  of,  this  species. 


Philip  Hanson  Hiss  179 

(d)  That  other  organisms  occur  which  in  their  fermentations 
are  indistinguishable  from  pneumococci,  but  which  either  mor- 
phologically or  in  agglutination  reactions  show  a  variation  from 
this  type.  Some  of  these  are  probably  temporarily  or  perma- 
nently modified  pneumococci  or  Streptococcus  mucosus ;  others, 
it  may  be,  are  streptococci  of  types  which  it  has  not  heretofore 
been  possible  to  recognize  and  describe.  Some  of  these  organ- 
isms were  isolated  from  pneumonic  lungs  at  autopsy,  or  from 
some  internal  source  such  as  the  circulating  blood,  and  presum- 
ably have  long  resided  under  conditions  which  may  be  considered 
adverse,  thus  bringing  about  a  modification  of  their  morphology 
or  physiology.  This  is  supported  by  the  observation  that  pneu- 
mococci from  such  sources — perfectly  typical  morphologically 
and  in  fermentative  activities — are  apt  to  show  a  lessening  of 
their  ability  to  agglutinate. 

In  connection  with  the  other  problem,  the  frequency  of  oc- 
currence of  true  pneumococci  in  the  mouth  of  healthy  persons, 
our  own  investigations,  limited  to  the  study  of  the  mouths  of 
twenty- two  individuals,  have  given  the  following: 

In  a  series  of  fifteen  persons  from  each  of  whom  one  specimen 
of  saliva  only  was  examined,  typical  pneumococci  were  found 
in  seven  out  of  the  fifteen  specimens,  i.e.,  46.6  %.  In  the  case 
of  the  remaining  seven  individuals  of  our  series,  repeated  tests, 
.extending  over  weeks  and  months,  were  made,  and  the  pneu- 
mococcus  was  demonstrated  upon  one  or  more  occasions  in  the 
saliva  of  six  out  of  the  seven,  i.e.,  85.7  %. 

It  seems,  therefore,  more  than  probable  that  practically  every 
individual,  at  least  during  the  winter  season,  when  exposed  to 
environmental  conditions  such  as  those  existing  in  New  York 
City,  acts  as  the  host  at  some  time  or  other,  and  probably  at  re- 
peated intervals,  of  organisms  of  the  true  pneumococcus  type. 

None  of  the  supposedly  normal  individuals  examined  by  us 
had  had  a  recognized  pneumococcus  infection  or  "cold"  within 
a  recent  time,  nor,  with  two  well-marked  exceptions,  did  any 
symptoms  of  infection  develop  in  those  whose  mouths  were 
found  to  contain  pneumococci. 

These  two  exceptions,  detailed  in  an  earlier  section,  strongly 


180        Oomparative  Stvdy  of  Pneumococci  and  Allied  Organisms 

suggest  the  etiological  relations  of  pneumococci  to  some,  at  least, 
of  the  "common  colds." 

In  conclusion,  it  is  a  pleasure  to  thank  the  various  investiga- 
tors connected  with  the  work  of  the  Commission,  for  their 
courtesy  in  furnishing  us  with  the  cultures,  upon  a  study  of 
which  any  success  our  work  may  have  attained  has  so  largely 
depended. 

REFERENCES. 

1.  Hiss.  "A  Contribution  to  the  Physiological  Differentiation  of  Pneumo- 
coccus  and  Streptococcus,  and  to  Methods  of  Staining  Capsules."  Jour.  Exp. 
Med.,  1905,  vi,  317;  abstract  in  Ctbl.  f.^'Bakt.,  1902,  xxxi,  302. 

2.  Hiss.  "A  Method  of  Obtaining  Mass  Cultures  of  Bacteria  for  Inocula- 
tion and  for  Agglutination  Tests ;  with  Special  Reference  to  Pneumococci  and 
Streptococci."     Jour.  Exp.  Med.,  1905,  vii,  2,  p.  223. 

3.  SchottmuUer.     Munch,  med.  Wochenschrift,  1903,  i,  909. 

4.  Rosenow.     Journ.  of  Infectious  Diseases,  1904,  i,  308. 

EXPLANATION  OF  THE  TABLE. 

The  table  contains  a  list  of  the  organisms  examined,  and  an  outline  record  of 
the  results  of  the  morphological  examinations  and  of  the  principal  fermenta- 
tion and  agglutination  tests.  Where  two  descriptions  occur  under  the  same 
heading,  as  in  the  morphology  and  inulin  fermentation  columns,  the  first  de- 
scription is  the  one  reported  to  us,  the  second  is  the  result  of  our  own  tests. 

The  sera  used  for  the  agglutination  tests  were  obtained  from  immunized  rabbits. 

The  agglutination  tests,  recorded  in  the  pneumococcus  immune  serum 
column,  were  not  all  made  in  the  same  serum.  The  letter  preceding  the  numer- 
als indicates  the  special  serum  in  which  the  test  was  made.  The  agglutination 
of  the  homologous  organisms  in  these  were  as  follows :  a — 400-800 ;  b — 400-800 ; 
c — 200—800;   d — 200—400. 

The  results  given  in  the  column  should  not,  therefore,  be  compared  with  each 
other,  except  when  preceded  by  the  same  letter,  but  with  the  agglutination  of 
the  homologous  organism. 

The  other  agglutination  tests  were  made  in  sera  from  Streptococcus  pyogenes 
and  Streptococcus  mucosus  immune  rabbits,  and  in  the  case  of  sera  "S.  7  "  and 
"CD  No.  3  "  from  rabbits  immunized  against  organismis  which  fermented  inulin 
but  were  atypical  morphologically  and  did  not  agglutinate  to  an  appreciable 
degree  in  pneumococcus  immune  sera.  The  P.  &  S.  "8"  serum  was  from  a 
rabbit  immunized  against  P.  &  S.  culture  "  8, "  which  is  a  non-inulin-fermenting 
capsulated  streptococcus. 

When  two  numbers  are  used  to  record  the  results  of  an  agglutination  test, 
the  first  indicates  the  highest  dilution  of  the  serum  in  which  a  complete  pre- 
cipitation of  the  agglutinated  organisms  occurred,  and  the  second  the  highest 
reading  with  a  hand  lens. 

All  agglutination  tests  were  made  in  small  test-tubes  by  the  macroscopic 
method,  and  careful  comparisons  were  always  made  with  the  normal  rabbit- 
serum  and  salt-solution  controls. 


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189 


THE  VIABILITY  OF  THE  PNEUMOCOCCUS  AFTER  DRY- 
ING:   A  STUDY  OF  ONE  OF  THE  FACTORS 
IN  PNEUMONIC  INFECTION. 

By  FRANCIS  CARTER  WOOD,  M.D.,  of  New  York, 

Adjunct  Professor  of   Clinical   Pathology,  College  of   Physicians   and  Surgeons, 
Columbia  University;  Pathologist  to  St.  Luke's  Hospital. 

{A  Study  from  the  Department  of  Pathology,  Columbia  University,  under  a  Grant 

from  the  Comm-ission  for  the  Investigation  of  Acute  Respiratory  Diseases, 

of  the  Departm,ent  of  Health  of  the  City  of  New  York.) 

The  exact  way  in  which  the  pneumococcus  reaches  the  lungs  of 
persons  suffering  from  pneumonia  due  to  that  organism  is  not  yet 
thoroughly  understood.  A  number  of  possibihties  have  been 
considered  which  may  be  indicated  as  follows : 

(i)  The  first  is  that  the  pneumococcus  is  frequently  present 
in  the  sahva,  and  that  when  the  resistance  of  a  person  carrying 
these  organisms  is  reduced,  for  example  by  exposure  or  overwork, 
an  infection  of  the  lungs  takes  place  either  by  extension  along 
the  tracheal  mucosa  or  by  the  direct  aspiration  into  the  lung  of 
particles  of  the  salivary  secretion  carrying  the  germs  with  them.^ 

(2)  A  second  possibility  which  may  be  considered  is  that  the 
pneumococcus  is  transferred  from  the  oral  or  pharyngeal  mucosa 
to  the  lungs  by  the  lymphatics  or  through  the  blood. 

(3)  Another  possibility  is  that  the  pneumococcus,  which  is 
capable  of  living  in  masses  of  dry  sputiim  for  some  time,  is  dis- 
tributed in  the  form  of  dust  derived  from  the  dried  sputtim 
particles  and  that  these  particles  are  inhaled,  thus  giving  rise  to 
a  pulmonary  infection. 

(4)  A  fourth  suggestion  is  that  the  pneumococcus  is  carried 

1  For  phases  of  this  problem  which  cannot  be  considered  here,  see  Wadsworth, 
Am,erican  Jour,  of  the  Med.  Sciences,  1904,  cxxvii,  851.  Other  papers  on  the 
subject  are:  Nenninger,  Zeit.  f.  Hyg.,  1901,  xxxviii,  94;  Klipstein,  Zeit.  f.  klin. 
Med.,  1898,  xxxiv,  191;  Durck,  Deutsches  Arch.  f.  klin.  Med.,  1897,  Iviii,  368; 
Wandel,  ibid.,  1903,  Ixxviii,  i. 

190 


Francis  Carter  Wood  191 

directly  from  person  to  person  either  by  the  transfer  of  the  normal 
nasal  or  saHvary  fluids,  which  may  contain  pnenmococci,  by 
coughing  or  sneezing,  or  by  the  spraying  of  fine  particles  derived 
from  the  sputum  of  those  suffering  from  pneumonia  or  other 
acute  inflammations  of  the  air  passages,  by  the  same  mechanical 
processes,  and  that  the  spray  particles  thus  formed  carry  virulent 
organisms  to  the  lungs. 

The  present  study  is  devoted  to  a  consideration  of  the  possi- 
bilities of  the  aerial  transmission  of  the  pneumococcus  either  in 
the  form  of  sprayed  particles  or  as  dust  derived  from  dried 
sputum,  the  modes  of  infection  from  the  saliva  as  given  in  the 
first  and  second  paragraphs  not  coming  within  the  scope  of  this 
investigation. 

While  a  good  deal  of  work  has  been  done  by  Cornet,  Fliigge,  and 
others  in  determining  the  viability  of  the  tubercle  bacillus  and 
other  organisms  in  fine  spray  and  also  after  drying  and  subjection 
to  various  physical  agents,  but  Httle  attention  has  been  directed 
to  the  pneumococcus  except  when  dried  in  relatively  large  masses 
of  sputum.  Most  observers  have  considered  the  pneumococcus 
as  an  organism  incapable  of  Hving  for  any  considerable  time  when 
suspended  in  the  form  of  fine  spray. 

As  the  results  of  recent  studies  2  on  the  biology  of  the  pneu- 
mococcus have  rendered  the  identification  of  that  organism 
relatively  easy,  and  as  some  of  the  earlier  studies  on  the  viability 
were  carried  out  with  bacteria  which  may  or  may  not  have  been 
the  pneumococcus,  it  seemed  to  the  writer  that  a  revision  and 
extension  of  some  of  the  older  investigations  might  be  of  value 
in  deciding  some  points  in  the  mode  of  transmission  of  this 
organism  which,  though  important,  have  not  yet  been  cleared  up, 

HISTORICAL    RESUME. 

Before  proceeding  to  the  description  of  the  methods  and  results 
of  personal  experiments,  it  may  be  well  to  give  a  short  resume  of 
the  work  done  by  other  observers  on  the  general  question  of 
transmissibility  of  the  pneumococcus  from  infectious  material 
to  hiunan  beings. 

2  Hiss,  Jour,  of  Ex  per.  Med.,  1905   vi,  317. 


192  Viability  of  the  Pneumococcus  after  Drying 

Viability  of  Pneumococci  in  Dried  Sputum. — The  earlier  ex- 
periments to  determine  the  dangers  of  air  infection  by  the  pneu- 
mococcus were  conducted  with  the  idea  of  fixing  the  length  of 
time  during  which  the  organism  would  remain  virulent  for  rabbits 
or  mice  after  drying  sputum  in  bulk,  the  powdering  and  diffusion 
of  the  powder  by  air  currents  being  thought  to  be  the  means  of 
transmission. 

It  was  known  that  the  pneumococcus  died  very  rapidly  in 
many  of  the  ordinary  culture  media.  In  fact  it  was  pointed  out 
by  Kruse  and  Pansini  ^  that  some  varieties  of  bouillon  made 
from  meat  infusion  were  highly  bactericidal  to  the  pneumococcus. 
The  same  observers  found  that  in  body  fluids,  however,  for  ex- 
ample in  sealed  tubes  containing  pleuritic  exudate,  the  pneu- 
mococcus may  remain  aHve  for  more  than  a  year,  if  kept  in  a 
cool,  dark  place.  They  showed  that  in  moist  sputum  preserved 
at  15°  C.  the  life  of  the  organism  is  very  short,  usually  but  three 
or  four  days.  In  sputum  kept  within  a  few  degrees  of  o°C., 
however,  the  life  of  the  organism  is  much  longer,  and  while  the 
fluid  loses  in  a  few  days  much  of  its  virulence  for  mice,  yet  living 
pneumococci  can  be  demonstrated  for  at  least  six  weeks  under 
these  conditions. 

Drying  of  the  sputum  in  the  air  at  incubator  temperatures 
killed  the  organisms  qmckly,  though  Guarnieri  *  found  that  rapid 
drying  in  a  desiccator  at  37°  C  preserved  the  virulence  for 
rabbits  for  four  months. 

Patella  ^  noted  that  rapid  drying  over  sulphuric  acid  at  16° 
C.  or  38°  C.  killed  the  organisms  promptly,  while  slow  drying  at 
low  temperatures  enabled  them  to  live  for  some  time.  As  it  has 
been  shown  by  Kirstein  ^  that  sulphuric  acid  probably  gives  off 
a  small  quantity  of  sulphur  trioxide,  which  would  act  destruc- 
tively upon  any  organisms  with^which  it  might  come  into  contact, 
the  rapid  death  frequently  observed  when  the  pneumococcus  is 
desiccated  over  this  meditim  may  be  due  to  the  bactericidal 

3  Zeit.  f.  Hyg.,  1892,  xi,  279. 

■♦  Atti  della  R.  Accad.  med.  di  Roma,  1888,  iv,  97. 

'  Ibid.,  447. 

«  Zeit.  f.  Hyg.,  1902,  xxxix,  166. 


Francis  Carter  Wood  193 

action  of  the  acid.  Drying  over  calcium  chloride  and  phosphoric 
anhydride  does  not  destroy  bacteria  so  quickly  as  drying  over 
sulphuric  acid. 

Foa  and  Bordoni-Uffreduzzi  ^  dried  rabbit's  blood  containing 
pneumococci  on  watch  glasses  and  found  that  the  micro-organisms 
were  alive  and  virulent  after  forty-five  days.  Agar  tubes  inocu- 
lated from  organs  and  kept  for  sixty  days  showed  an  abundant 
growth  when  placed  in  the  incubator — an  evidence  that  the 
organisms  may  remain  alive  under  suitable  conditions.  At  the 
time  at  which  this  work  was  done,  however,  the  difference  between 
the  pneumococcus  and  the  meningococcus  had  not  been  thoroughly 
defined,  and  the  writers  termed  the  organism  with  which  they 
worked  a  meningococcus  because  it  was  obtained  from  a  case  of 
cerebrospinal  meningitis.  From  their  description  of  its  bio- 
logical features,  however,  the  organism  seems  to  have  been 
Diplococcus  pneumorrise.  It  was  positive  to  Gram,  showed  a 
capsule,  and  killed  rabbits  promptly.  Apparently  without 
recognizing  their  nature,  the  writers  figure  pneumococci  inclosed 
in  the  phagocytic  cells  of  the  pneumonic  exudate  of  the  rabbit. 

Five  years  later  Bordoni-Uffreduzzi  ^  reported  the  results 
obtained  by  contaminating  pieces  of  linen  with  pneumonic 
sputum.  The  cloth  was  allowed  to  dry  at  room  temperatures. 
In  one  specimen  exposed  to  diffuse  light  the  bacteria  remained 
alive  for  nineteen  days,  as  determined  by  injection  into  rabbits 
of  scrapings  from  the  cloth;  in  another,  fifty-five  days.  The 
sputum  dried  in  sunlight  was  virulent  after  twelve  hours.  The 
results  differ  somewhat  from  those  of  Patella,  possibly  depending 
upon  the  technique.  Patella  using  silk  threads  soaked  in  the 
blood  of  an  animal  dying  of  a  pneumococcus  septicaemia  or 
threads  soaked  in  broth  cultures  of  the  pneumococcus. 

The  results  of  Cassedebat  ^  differ  considerably  from  those  of 
Bordoni-Uffreduzzi.  The  experiments  were  conducted  as  fol- 
lows: SputiuTL  was  tested  for  its  virulence  and  found  to  kill 
rabbits.     Specimens  of  this  pneumonic  sputum  were  then  dried 

iZeit.  f.  Hyg.,  1888,  iv,  67. 

8  Arch.  p.  I.  sc.  med.,  1891,  xv,  341. 

9  Revue  d'Hygiene,  1895,  xvii,  1066. 


194  Viability  of  the  Pneumococcus  after  Drying 

on  cloth  in  the  air  but  protected  from  the  direct  rays  of  the  sun. 
Fragments  of  the  cloth  were  soaked  in  water  and  the  fluid  in- 
jected into  a  rabbit.  The  results  showed  that  the  dried  sputum 
killed  rabbits  at  periods  varying  from  five  to  twenty-six  days, 
and  that  fresh  sputum  from  the  eighth  and  ninth  days  of  the 
disease  would  not  kill  rabbits.  Apparently  the  writer  reHed  on 
the  gross  post-mortem  findings  for  the  identification  of  the 
pneumoCoccus.  There  is  no  mention  of  morphological  studies 
of  the  blood  to  identify  capsulated  organisms  or  attempts  to 
cultivate  the  pneumococci.  The  results  have,  therefore,  but 
sHght  value. 

Ottolenghi^o  repeated  the  studies  of  Bordoni-Uffreduzzi  with 
the  following  results.  The  experiments  were  carried  out  with 
three  specimens  of  pneumonic  sputiim  from  the  fourth  or  fifth 
day  of  the  disease.  The  sputum  was  spread  on  linen  cloth  and 
allowed  to  dry  in  diffuse  light  at  a  temperature  of  15°  C.  to  20°  C. 
In  explaining  the  results  which  he  obtained,  the  author  calls 
attention  to  the  fact  that  the  inoculation  of  the  material  into  a 
rabbit  is  not  sufficient  to  determine  whether  the  pneumococcus 
is  dead  or  not.  The  death  of  the  rabbit  merely  determines  the 
presence  of  organisms  virulent  for  that  animal,  but  non- virulent 
forms  may  be  present.  He  therefore  made  cultures  from 
the  sputimi  at  the  same  time  that  he  carried  out  the  animal 
inoculations.  The  first  specimen  tested  lost  its  virulence  for 
animals  thirty-six  days  after  the  preparation  was  made,  whereas 
pneumococci  could  be  obtained  culturally  for  sixty  days  after 
drying.  In  the  second  specimen,  both  methods  showed  pneu- 
mococci at  the  end  of  seventy  days,  and  from  the  third  specimen 
pneumococci  were  isolated  on  the  eighty-third  day.  On  the 
basis  of  these  experimients,  the  author  considered  that  Diplococ- 
cus  lanceolatus  can  retain  its  virulence  in  dried  sputum  for  at 
least  twenty  days,  and  that  it  remains  alive  for  a  considerable 
time  after  the  virulence  has  disappeared.  The  virulence  per- 
sisted longest  in  a  thin,  frothy  sputum. 

In  some  experiments  recently  reported  by  Heim,i^  the  viability 

>o  Cent.  f.  Bakt.,  1899,  xxv,  Abt  i,  120. 
11  Zeit.  f.  Hyg.,  1905,  I,  123. 


Francis  Carter  Wood  195 

of  the  pneumococcus  after  drying  was  much  greater  than  has 
usually  been  assumed.  Silk  threads  were  dipped  into  the  heart's 
blood  of  cats,  rabbits,  and  mice,  which  had  been  killed  by  injec- 
tions of  pneumococci.  The  threads  were  dried  in  a  desiccator 
over  calcium  chloride,  and  were  then  removed  at  various  periods, 
placed  in  bouillon  or  agar,  and  the  resulting  culture  inoculated 
into  mice.  The  organisms  were  virulent  in  some  cases  after  487 
days.  Great  variations,  however,  were  observed.  Some  of  the 
cultures  from  the  threads  no  longer  gave  rise  to  septicaemia  after 
sixty-six  days.  One  case  was  not  virulent  after  nine  days.  In 
empyema  pus  the  pneumococcus  remained  virulent  for  377  days. 
Another  specimen  of  empyema  pus  contained  organisms  which 
Heim  states  lie  between  the  pneumococcus  and  the  streptococcus 
groups.  These  were  virulent  at  the  end  of  149  days  and  con- 
tained viable  organisms  at  the  end  of  383  days.  The  conditions 
of  these  experiments  are,  however,  highly  artificial  and  cannot  be 
considered  as  applying  very  definitely  to  the  question  of  aerial 
infections.  The  alternate  drying  and  moistening  of  the  organ- 
isms due  to  the  varying  amounts  of  moisture  in  the  air  of  rooms 
is  very  important  as  determining  the  rapid  death  of  the  pneu- 
mococcus, while  protection  from  such  changes  by  sealing  the 
substances  carrying  the  bacteria  in  vessels  containing  calcium 
chloride  tends  to  prolong  the  life  of  the  parasite. 

Mode  of  Distribution  of  Dried  Sputum  Particles. — It  thus 
having  been  conclusively  shown  that  the  pneumococcus  can  re- 
main alive  for  a  considerable  length  of  time  in  dried  sputum,  it  is 
necessary  to  demonstrate  that  this  dried  sputum,  which  under 
ordinary  conditions  is  firmly  adherent  to  the  substance  on  which 
it  is  dried,  can  in  some  way  be  reduced  to  a  powder  and  thus 
inhaled.  Such  conditions  can  only  be  realized  when  the  sputum 
is  dried  in  handkerchiefs,  bedding,  or  clothing,  and  the  contami- 
nated material  handled,  or  when  the  sputum  is  deposited  upon  the 
floor  and  pulverized  by  persons  walking  over  the  infected  area,  or 
distributed  in  the  air  by  dry  sweeping  of  the  floor,  or  brushing 
of  infected  clothes,  etc.  This  mode  of  distribution  of  infectious 
material  has  been  studied  chiefly  in  connection  with  the  tubercle 
bacillus,  because  of  the  ease  of  identifying  that  organism  in  the 


196  Viability  of  the  Pneumococcus  after  Drying 

infectious  dust,  and  the  difficulty  attendant  upon  the  recognition 
of  the  pneumococcus  under  the  same  conditions.  The  results 
obtained,  however,  can  be  legitimately  transferred  to  the  pneu- 
mococcus, leaving  out  of  the  question  for  the  moment  the 
viability  of  the  latter  organism  after  drying.  Much  of  our 
knowledge  on  this  subject  we  owe  to  the  studies  of  Comet  and  of 
Flugge  and  his  pupils. 

Some  of  the  results  which  have  been  obtained  are  as  follows: 
Comet,  ^2  -^7]2o  considers  this  dust  inhalation  the  most  important 
means  of  infection  in  tuberculosis,  has  demonstrated  the  infectious 
nature  of  the  dust  of  rooms  in  which  persons  suffering  from  tuber- 
culosis had  lived,  and  showed  that  the  risk  of  infection  depended 
very  largely  upon  the  expectoration  of  the  sputum  on  handker- 
chiefs, bedding,  carpets,  or  clothing,  and  the  subsequent  drying 
of  the  fluid.  He  considered  that  there  was  practically  no  danger 
of  direct  infection  in  tuberculosis  by  particles  of  sputtim  expelled 
by  coughing,  but  that  the  sputtmi  expectorated  in  large  masses 
and  dried  on  the  bedding  or  floor  was  the  chief  source  of  the  dis- 
ease. His  results  have  been  disputed  b}^  Frankel  and  also  by 
Flugge  and  his  pupils,  who  have  shown  that  it  is  difficult  to 
pulverize  the  sputum  to  a  sufficient  degree  to  produce  a  powder 
fine  enough  to  be  carried  by  air  currents  of  moderate  velocity  or 
to  remain  long  in  suspension. 

Sticher,^^  in  order  to  test  this,  pulverized  dried  tubercu- 
lous sputum  and  found  that  while  the  particles  could  be  carried 
by  a  current  of  air  with  a  velocity  of  i  cm.  per  second  to  a 
height  of  one  meter,  3'et  the  number  of  bacteria  which  even  the 
very  fine  dust  particles  carried  was  small,  and  hence  infection  was 
not  likely  to  result  from  the  dissemination  of  such  dust.  The  air 
currents  in  rooms  without  special  ventilation  rareh^  exceed  one 
centimeter  per  second  and  do  not  transport  for  any  length  of 
time  coarse  dust  such  as  is  produced  b}'  powdering  sputum. 

Beninde  ^"^  repeated  the  experiments  of  Sticher  and  found  that 

1 2  Die  Tuhercnlose,  KothnageVs  Spec.  Path.  u.  Therap.,  Bd.  xiv,  p.  209.  Vienna, 
1899. 

»3  Zeit.  f.  Hyg.,  1899,  xxx,  163. 
1*  Ibid.,  193. 


Francis  Carter  Wood  197 

using  handkerchiefs  contaminated  with  tuberculous  sputum  it 
was  impossible  while  the  latter  was  still  damp  to  remove  any 
bacilli  from  the  surface  of  the  cloth  by  a  stream  of  air  with  a 
velocity  of  lo  cm.  per  second,  this  being  the  upper  limit  of  air 
currents  in  well  ventilated  rooms.  Tubercle  bacilli,  however, 
could  be  removed  by  using  an  air  stream  of  i  cm.  per  second, 
but  only  after  the  handkerchief  had  been  carried  about  for  two 
days  and  was  thoroughly  dry. 

Further  investigations  in  this  hne  were  made  by  Neisser,^^ 
who  used  pneumonic  sputum  and  studied  the  conditions  obtained 
by  mixing  the  fluid  with  dust  and  drying.  This  combination  was 
then  finely  pulverized  and  carried  from  one  chamber  to  another 
by  an  air  current  with  a  speed  of  from  2.8  mm.  per  second  to 
23  cm.  per  second.  As  a  control,  some  of  the  sputum  used  was 
injected  into  mice  and  shown  to  be  virulent.  As  soon  as  pneu- 
monic sputum  and  the  dust  mixture  dried,  it  would  no  longer 
kill  mice,  therefore  the  danger  of  dust  infection  by  inhaling 
pulverized  and  dried  sputum  seemed  exceedingly  remote. 

It  should  be  repeated,  however,  that  while  it  is  not  difficult 
to  obtain  dry  and  finely  pulverized  pneumonic  sputum  under 
experimental  conditions,  yet  practically  the  drying  of  the  mass 
is  rarely  complete  enough  to  permit  thorough  powdering,  and 
the  particles  which  are  removed  mechanically  from  the  sputum, 
after  this  fluid  has  dried  on  cloth,  wood,  or  metal,  are  of  such 
dimensions  that  they  cannot  be  carried  for  any  considerable 
distance  by  the  air  currents  ordinarily  found  in  well  ventilated 
rooms,  or,  if  so  carried,  remain  in  suspension  for  a  very  short 
time.  The  contaminated  particles  are  not  likely,  therefore,  to 
be  inhaled  even  by  persons  in  close  contact  with  the  patient, 
and  are  still  more  unlikely  to  lead  to  an  infection  of  persons  in 
other  rooms  or  at  a  distance.  Only  in  structures  subject  to 
strong  draughts,  such  as  factories  or  railroad  carriages,  are  air 
currents  likely  to  be  strong  enough  to  render  these  coarse  particles 
dangerous.  The  possibilities  of  infection  are  reduced  to  a  mini- 
mum when  the  dust  particles  are  blown  about  in  the  open  air. 
The    dilution    is    so    great    and    the    death    of    the  organisms 

•5  Zeit.  f.  Hyg.,  1898,  xxvii,  175. 


198  Viability  of  the  Pneumococcus  after  Dryiing 

contained  in  the  dust  is  so  rapid  that  infection  cannot  be  as- 
sumed as  likely  to  occur. 

Gemiano  ^^  studied  the  effect  of  drying  the  pneumococcus  with 
dust  by  a  different  technique.  The  results  which  he  obtained 
with  cultures  of  the  pneumococcus  showed  that  when  mixed 
with  sterile  dust  and  dried,  the  organisms  died  within  two  days, 
imless  the  drying  took  place  at  a  temperature  below  o°  C, 
when  the  organisms  remained  virulent  for  eight  days.  Mixed 
with  sand  and  vegetable  soil,  the  organisms  died  in  two  days; 
mixed  with  volcanic  ash  (Tuffboden),  and  kept  moist,  the  organ- 
isms remained  aUve  for  six  days. 

Another  group  of  experiments  with  another  organism,  pre- 
sumably pneumococcus,  showed  a  very  considerable  variation 
from  the  above.  The  cultiure  mixed  with  brick  dust  remained 
infectious  for  forty  days,  either  kept  moist,  dried  in  the  air,  or 
dried  over  sulphuric  acid.  Mixed  with  sand  this  same  organism 
was  infectious  for  sixty  days  when  kept  moist  or  dried  at  room 
temperature,  and  for  fifty  days  when  dried  over  sulphuric  acid. 
This  organism  was  obtained  during  an  epidemic  of  pneimionia 
which  occurred  in  a  small  village,  a  number  of  cases  developing 
about  the  same  time.  Germano  thinks  that  possibly  the  epi- 
demic was  due  to  the  long  life  of  this  organism  in  the  air. 

The  results  of  experiments  by  the  same  writer  with  pneumonic 
sputum  confirmed  the  facts  which  have  been  observed  as  to  the 
long  life  of  the  bacteria  when  dried  in  coarse  particles.  For 
example,  pneumonic  sputum  mixed  with  room  dust  kept  moist 
was  virulent  for  twelve  days,  kept  dry,  for  twenty  days,  dried 
over  sulphuric  acid,  for  sixteen  days,  dried  at  a  low  temperature, 
for  eight  days.  The  last  results  are  thus  somewhat  different 
from  those  obtained  by  Patella  and  Neisser.  Sputum  mixed 
with  earth  (Humusboden)  was  virulent  for  twelve  days  when 
kept  moist,  for  one  hundred  and  forty  days  when  dried  in  the 
air,  and  for  one  htmdred  days  when  dried  over  sulphuric  acid. 
With  a  low  temperature  the  virulence  was  retained  for  only 
sixteen  days. 

The  writer  considers  that  slight  variations  in  the  type  of  the 

^(•Zeit  f.  Hyg.,  1897,  xxv,  439;  ibid.,  1897,  xxvi,  66  and  273. 


Francis  Carter  Wood  .  199 

diplococcus  may  contribute  very  largely  to  the  length  of  time 
during  which  the  organisms  can  resist  drying.  It  was  cer- 
tainly proven  that  they  remained  virulent  longer  when  dried 
than  when  kept  moist.  At  low  temperatures  the  short  life 
of  the  organisms  seemed  to  be  conditioned  by  the  fact  that  the 
sputtun  dried  slowly  at  a  point  near  o°  C.  so  that  the  bacteria 
were  really  kept  moist.  The  rapidity  of  the  drying  process  at 
room  temperature  had  no  influence  upon  the  life  of  the  diplococ- 
cus. Germano  concludes,  finally,  that  air  infection  of  human 
beings  by  the  organism  is  possible  with  the  pneumococcus,  but 
the  chances  are  relatively  small. 

It  will  be  seen  from  the  preceding  resume  that  the  views  ex- 
pressed by  Cornet  on  the  possibility  of  dust  infection  in  tubercu- 
losis can  hardly  be  considered  as  of  great  import  in  pneumonia. 
The  sputum  of  pneumonic  patients  is  often  exceedingly  viscid  and 
thick;  it  is  not  usually  produced  in  the  same  abundance  as  in 
pulmonar}'  tuberculosis;  and  the  coughing  of  the  patients  is 
rarely  so  prolonged  or  strongly  expulsive.  Coarse  particles  are 
therefore  less  Hkely  to  be  distributed  in  the  neighborhood  of  such 
a  patient.  When  such  particles  are  expelled,  the  results  of 
Sticher  and  Beninde  show  that  the  air  currents  which  are  ordi- 
narily present  in  well  ventilated  houses  are  insufficient  to  remove 
bacteria  from  the  moist  or  dried  sputum.  The  risk  of  infection 
must  be  largely  confined  to  those  who  handle  the  bedding,  etc., 
of  pneumonic  patients. ^^  The  experiments  of  Neisser  and 
Germano  are  not  wholly  consistent,  but  tend  to  show  that  the 
pneumococcus  dies  early  when  dried  in  finely  divided  sputum. 
It  is  possible  that  in  some  of  Germano 's  experiments  the  organ- 
ism was  not  the  one  which  we  are  accustomed  to  consider  as  the 
pneumococcus . 

Conditions  under  which  the  Pneumococctts  may  he  Transmitted 
in  Sprayed  Particles. — We  are  largely  indebted  to  Fliigge  and  his 
co-workers  for  the  experimental  investigation  of  the  theory  that 
the  transfer  of  pathogenic  bacteria  from  one  person  to  another  is 

17  See  in  this  connection  a  report  by  Edson  and  Ghriskey  on  "A  Hospital 
Epidemic  of  Pneumococcus  Infections,"  Trans,  of  the  Philadelphia  Coll.  of  Phys., 
1904,  xxvi,  6. 


200  Viability  of  the  Paeuraococcus  after  Drying 

possible  by  the  aerial  transmission  of  fine  spray  particles.  They 
considered  that  the  spraying  of  fine  particles  of  sputum  of  saliva 
by  talking,  coughing,  sneezing,  or  spitting,  might  carry  infec- 
tious material  from  one  person  to  another  if  the  specific  organism 
remains  alive  in  such  spray  for  a  sufficient  time  to  permit  the 
floating  particles  to  be  carried  from  the  patient  to  other  persons 
in  the  vicinity  by  the  air  currents  produced  for  purposes  of 
ventilatioti. 

The  studies  of  this  means  of  transmission  of  the  pneumococcus 
are  not  numerous,  and  we  have  obtained  a  large  part  of  our 
knowledge  concerning  such  transmission  by  investigating  the 
conditions  of  infection  which  obtain  in  connection  with  pul- 
monary tuberculosis.  Here  again  most  of  the  factors  are  the 
same  with  both  the  pneumococcus  and  the  tubercle  bacillus,  and 
we  can  without  impropriety  transpose  the  results  obtained  by  the 
study  of  one  organism  to  another. 

While  it  has  been  repeatedly  shown  that  the  air  expired  from 
the  lungs  during  quiet  breathing  contains  no  bacteria,  yet  in  dis- 
ease a  small  number  of  bacteria  may  be  given  off.  It  has  been 
shown  by  Koelzer  ^^  that  tuberculous  persons  by  a  sort  of  internal 
spraying  give  off  tubercle  bacilli  even  during  quiet  breathing,  and 
that  the  fine  drops  containing  tubercle  bacilh  are  produced  by  the 
pulverizing  of  the  thick  mucus  in  the  bronchi  during  the  passage 
of  the  expiratory  current — the  same  physical  condition  which 
gives  rise  to  the  rales  heard  on  auscultation  of  the  chest.  Laryn- 
geal tuberculosis  increases  the  liability  to  the  contamination  of 
the  expired  air.  Koelzer  obtained  positive  results  in  one  case 
out  of  fifteen  persons  examined,  care  being  taken  to  see  that  no 
coughing  took  place  while  the  Petri  dishes  were  exposed  to  the 
expiratory  current.  A  similar  observation  is  recorded  by 
Schaffer,^^  -^^j^^q  was  able  to  demonstrate  lepra  bacilli  in  the  ex- 
pired air  of  persons  suffering  from  lesions  of  the  nose  and  throat 
due  to  that  organism. 

The  results  of  many  observations  on  the  bacterial  content  of 
the  expired  air  have  shown,  however,  that  this  source  of  infection 

18  Zeit.  f.  Hyg.,  1903,  xliv,  217. 

>9  Arch.  f.  Derm.  u.  Syphilis.  1898,  xliii  and  xliv,  159. 


Francis  Carter  Wood  201 

may  be  practically  neglected.  On  the  other  hand,  coughing  and 
especially  sneezing,  as  previously  mentioned,  cause  an  abundant 
spraying  of  fine  fluid  particles  which  may  contain  bacteria. 

Koeniger  20  has  studied  with  great  care  the  conditions  under 
which  this  spraying  takes  place.  Large  numbers  of  particles 
are  produced  when  the  expiratory  stream  is  interrupted  and 
then  suddenly  begun.  The  drops  are  formed  in  the  portion  of 
the  respiratory  tract  where  the  stoppage  of  the  air  current  takes 
place.  Particles  produced  in  the  larynx  are  often  stopped  by 
the  lips  so  that  the  method  which  the  person  uses  in  coughing 
causes  great  variations  in  the  niunber  of  particles  expired  in  the 
air.  If  the  mouth  be  kept  nearly  closed,  the  laryngeal  particles 
do  not  escape  in  large  nimibers.  If,  however,  the  mouth  is 
tightly  closed,  drops  may  be  produced  from  the  passage  of  the  air 
currents  over  the  lips,  as  the  latter  are  pushed  open  by  the 
cough.  Particles  are  also  sprayed  out  in  clearing  the  throat. 
Loud  speaking  gives  more  spraying  than  quiet  conversation,  and 
the  method  of  articulation  exerts  considerable  influence.  The 
letters  k,  p,  f,  and  t  cause  more  spraying  than  vowels  or  other 
consonants.  It  is  probable  that  more  germs  are  sprayed  from  a 
thin,  watery  sputum  than  from  a  thick,  mucous  variety,  but  the 
intensity  of  the  coughing  impulse  is  much  more  important  than 
the  consistence  of  the  sputum.  More  particles  are  expelled 
when  the  cough  is  short  and  sharp.  Koeniger 's  studies  on 
spraying  were  made  by  infecting  the  mouth  of  the  experimenter 
with  B.  prodigiosus  and  other  saprophytic  organisms  and  then 
exposing  large  numbers  of  plates  while  talking,  coughing,  or 
sneezing. 

Dimensions  of  the  Sprayed  Particles. — The  size  of  the  particles 
produced  by  coughing  varies  greatly.  Heymann  21  studied  the 
size  and  number  of  the  drops  by  catching  the  spray  produced  by 
coughing  on  glass  slides  and  measuring  the  size  of  the  drops  so 
produced.  The  finer  particles  had  a  diameter  when  flattened  on 
glass  of  from  thirty  to  forty  micra.  Sneezing,  according  to  my 
own  observations,  may  give  rise  to  a  very  fine  spray,  the  particles 
not  measuring  over  ten  to  twenty  micra.     The  same  is  true  of  the 

20  Zeit.  f.  Hyg.,  1900,  xxxiv,  119.  21  Ibid.,  1899,  xxx,  139. 


202  Viability  of  the  Pneumococcus  after  Drying 

drops  produced  in  a  spray  apparatus  using  either  a  hand  bulb  or 
air  at  high  pressure.  Many  of  the  particles  are  very  small  and 
contain  no  bacteria.  Those  above  twenty  micra  usually  contain 
one  or  more  organisms  if  the  sputum  is  rich  in  bacteria.  Even 
the  smaller  particles,  however,  though  they  may  not  contain 
bacteria,  when  collected  and  examined  are  found  to  have  a 
nucleus  formed  either  by  mucus  or  by  salt  crystals.  The  evapora- 
tion from,  these  small  particles  when  sprayed  into  the  air  is  ex- 
ceedingly rapid,  because  of  their  small  size. 22 

Spatial  Distribution  of  the  Sprayed  Particles. — Fliigge  in  a 
series  of  papers  23  has  shown  that  a  person  with  a  cough  sprays 
fine  particles  into  the  surrounding  air,  the  radius  of  the  zone  of 
such  spraying  being  usually  one  meter,  rarely  two  meters. 
Within  this  area  therefore  the  air  may  contain  floating  particles 
carrying  pathogenic  bacteria.  Flugge  has  shown  that  fine  dust 
particles  laden  with  bacteria  may  be  carried  horizontally  by  a 
stream  of  0.2  mm.  per  second,  one  five-hundredth  of  the  speed 
of  a  barely  perceptible  draught.  Upward  translation  of  these 
motes  requires  a  shghtly  greater  wind  velocity,  about  0.3  to  0.4 
mm.  per  second.  Stronger  currents  of  air  may  carry  them  to 
great  distances.  Hutchinson  2*  was  able  to  demonstrate  the 
transportation  of  particles  containing  B.  prodigiosus  for  a  dis- 
tance of  600  meters.  The  drops  produced  by  coughing,  sneezing, 
etc.,  are  usually  larger  and  heavier  than  those  just  mentioned, 
and  Heymann  25  has  shown  that  a  large  proportion  of  them  settle 
out  of  the  air  of  an  ordinarily  ventilated  room  within  an  hotu-. 
Particles  of  this  size  are  not  transported  laterally  to  any  very 
great  extent.  In  fact,  it  is  exceedingly  difficult  to  demon- 
strate tubercle  bacilli  in  the  air  of  wards  containing  tuberculous 
patients. 

As  the  sprayed  particles  settle  they  adhere  to  the  furniture, 
walls,  bedding,  and  carpets,  and  dry.  It  is  then  impossible  to 
remove  them  by  any  stream  of  air  within  the  limits  of  ordinary 

22  See  on  this  point  Thomson,  Conduction  of  Electricity  through  Gases,  Cam- 
bridge, 1903,  p.  135. 

23  Zeit.  f.  Hyg.,  1897,  xxv,  179;   tbid.,  1899,  xxx,  107;   ibid.,  1901,   xxxviii,  i. 
^^  Ibid.,  1 90 1,  xxxvi,  223. 

2  5  Ibid.,  xxxviii,  21. 


Francis  Carter  Wood  203 

ventilation  currents.  It  is  possible  to  remove  these  particles, 
however,  by  dry  brushing,  sweeping,  or  dusting,  and  the  powder 
so  formed  may  float  for  a  long  time  in  the  room  or  be  transported 
to  adjacent  ones. 

Bacterial  Content  of  Sprayed  Particles. — Direct  evidence  of 
"the  bacterial  content  of  the  sprayed  sputum  has  been  obtained 
by  B.  Frankel,  who  examined  the  contents  of  two  hundred  and 
nineteen  face  masks  each  of  which  had  been  worn  for  twenty-four 
hours  by  persons  whose  sputum  had  contained  tubercle  bacilli. 
In  twenty-six  of  these  masks  tubercle  bacilli  could  be  demon- 
strated. Frankel  assumes  that  in  thirty- two  days  2600  tubercle 
bacilli  had  been  caught  in  masks  which  would  otherwise  have 
escaped  into  the  air.  In  a  considerable  number  of  cases  of 
tuberculosis,  however,  Frankel  pointed  out  that  the  masks  re- 
mained uninfected  so  that  only  a  small  number  of  patients  could 
be  shown  to  cough  out  drops  of  sputum  or  saliva  containing 
tubercle  bacilh. 

The  presence  of  virulent  organisms  in  sprayed  sputum  has  been 
verified  by  other  observers. 2 ^  The  most  detailed  study  is  per- 
haps that  of  Heymann,27  who  examined  with  especial  care  the 
conditions  attending  the  spraying  of  tuberculous  sputum  by 
patients  under,  so  to  speak,  natural  conditions — that  is,  the 
coughing  was  not  forced,  the  patients  simply  being  confined  to  a 
room  while  the  tests  were  carried  out.  The  particles  sprayed 
out  by  the  patients  were  collected  and  found  to  contain  abundant 
tubercle  bacilh. 

An  interesting  example  of  the  large  numbers  of  bacteria  which 
may  be  expelled  is  that  reported  by  Schaffer  28  where  one  leprous 
patient  at  a  single  sneeze  gave  off  25,000  bacilli,  and  another 
patient,  110,000.  Patients  with  severe  lesions  of  the  tuberculous 
type  gave  off  from  10,000  to  185,000  lepra  bacilli  in  ten  minutes' 
talking.  The  sprayed  bacteria  were  caught  on  sHdes  placed 
close  to  the  patients'  mouths  and  only  a  very  few  could  be 
demonstrated  at  a  distance  of  one  and  one-half  meters. 

26  V.  Hubener,  Zeit.  f.  Hyg.,  1898,  xxviii,  348;  v.  Weismayer,  Wiener  klin. 
Woch.,  1896,  p.  1039;  Bing,  Zent.  f.  innere  Medizin,  1905,  p.  54;  Mendes  de 
Leon,  Arch.  f.  klin.  Chir.,  1904,  Ixxii. 

2'  Zeit.  f.  Hyg.,  1901,  xxxviii,  21. 
8  Arch.  f.  Derm.  u.  Syphilis,  1898,  xliii  and  xliv,  159. 


204  Yiability  of  the  Pneumococcus  after  Drying 

Hamilton  ^^  finds  ttiat  streptococci  are  expelled  from,  the 
mouth  by  coughing  or  even  by  breathing  by  persons  with  strepto- 
coccus infection  of  the  upper  air  passages. 

Life  of  the  Bacteria  in  the  Spray  Particles. — In  order  to  study 
the  length  of  life  of  bacteria  in  sprays,  Laschtschenko  ^^  atomized 
diluted  pneumonic  sputum  (ten  parts  sputum  and  from  one  to 
two  parts  water)  in  a  closed  vessel  and  caused  the  particles  to 
be  carried  upwards  for  one  meter  by  a  current  of  air  of  from 
6  to  lo  mm.  per  second. 

The  spraying  was  continued  under  low  pressure  for  one  and 
one-half  hours.  The  particles  were  collected  and  the  fluid 
injected  into  mice,  with  three  positive  and  two  negative  results. 
Spraying  undiluted  sputum  with  air  speeds  of  lo  to  12  mm.,  he 
obtained  one  positive  and  six  negative  results,  the  infectious 
nature  of  the  sputum  being  previously  determined  by  injecting 
mice.  Using  the  same  apparatus  and  conditions  with  phthisical 
sputum,  a  positive  result  was  obtained  in  all  cases  with  air  speeds 
of  from  6  to  14  mm.  per  second,  the  sputum  being  diluted  and 
undiluted.  The  spray  was  produced  by  a  very  low  air-pressure 
stream.  The  results  show  that  the  pneumococcus  and  the 
tubercle  bacillus  can  live  for  a  longer  or  shorter  time  in  sprayed 
sputum.  The  writer  gives  no  explanation  of  the  fact  that  many 
more  positive  results  were  obtained  with  tuberculous  sputum 
than  with  the  pneumococcus,  but  it  is  evident  from  my  own 
experiments,  to  be  given  later,  that  the  drying  which  the  pneu- 
mococci  underwent  while  carried  up  in  the  air  current  was 
sufficient  to  kill  many  of  the  organisms. 

A  phenomenon  noted  by  Koeniger  ^^  is  of  interest  in  this  con- 
nection. He  observed  that  after  spraying  large  quantities  of 
cultures  of  B.  prodigiosus  over  the  floor  and  furniture  of  a  room 
it  was  impossible  to  obtain  colonies  of  this  organism  on  exposed 
plates  even  when  large  amounts  of  dust  were  produced  by  ener- 
getic brushing.  Growths  were  obtained  from  many  other 
organisms  but  not  from  the  prodigiosus.  This  fact  was  not 
fully  understood  by  Koeniger,  who  states  that  the  results  of  his 

29  Jour,  of  the  American  Med.  Assoc,  1905,  p.  1108. 

3°  Zeit.  f.  Hyg.,  1899,  xxx,  133.  3t  Ibid.,  1900,  xxxiv,  119 


Francis  Carter  Wood  _  205 

experiments  show  that  the  bacteria  must  be  moist  to  produce  a 
growth.  The  true  explanation  was  given  shortly  afterward  by 
Kirstein,  who  showed  that  the  reason  for  the  negative  results 
was  that  B.  prodigiosus  when  sprayed  in  fine  particles  dried 
rapidly  and  was  promptly  killed,  especially  when  exposed 
to  diffuse  daylight.  In  one  set  of  experiments  by  the  latter, 
B.  prodigiosus  was  sprayed  in  two  rooms  and  the  falling 
germs  caught  on  glass  plates.  In  the  dark  room  the  bacteria 
remained  alive  for  fifteen  days,  in  the  well-lighted  room  for  only 
three  days. 

Similar  results  were  obtained  by  Kirstein  22  for  pathogenic 
organisms  such  as  the  typhoid  bacillus,  which  remained  alive  for 
only  a  few  hours,  the  tubercle  bacillus,  which  was  alive  for  from 
four  to  eight  days  in  diffuse  light  and  as  long  as  forty  days  in  the 
dark. 

Staphylococcus  pyogenes  aureus  and  streptococcus  remained 
alive  for  from  ten  to  sixteen  days;  diphtheria  bacilli,  less  than 
twenty-one  hours;  anthrax  bacilli,  nearly  ten  weeks.  The 
pneumococcus  was  not  investigated,  as  the  author  assumed 
from  the  results  of  previous  studies  by  Neisser  and  others  that 
prompt  death  of  the  pneumococcus  occurred  after  drying. 

In  a  more  recent  paper,  Kirstein  ^^  finds  that  the  tubercle 
bacillus  lives  for  from  eight  to  fourteen  days  when  sputimi  is 
sprayed  on  fine  dust,  from  four  to  seven  days  when  tuberculous 
sputum  is  finely  powdered,  five  days  when  deposited  on  fine  cloth 
fibers,  and  finally  that  the  bacillus  lives  but  three  days  on  fine 
street  dust  although  it  was  alive  for  eight  days  on  coarse  dust  of 
the  same  variety.  In  all  of  these  tests  the  bacteria  were  ex- 
posed to  diffuse  daylight. 

All  observers  are  agreed  then  that  the  life  of  the  bacteria  when 
sprayed  and  dried  may  be  safely  assumed  to  be  much  shorter 
than  when  they  are  dried  in  masses.  Diffuse  light  and  especially 
sunlight  rapidly  destroy  the  organisms,  while  preser\^ation  in  a 
dark,  cool  place  tends  to  prolong  their  existence. 

32  Zeit.  f.  Hyg.,  1900,  xxxv,  123;  ibid.,  1902,  xxxix.  See  also  Ficker,  Zeit.  f. 
Hyg.,  1898,  xxix,  I. 

33  Zeit.  f.  Hyg.,  1905,  1,  186. 


206  Viability  of  the  Pneumococcits  after  Drying 

Summary. — As  will  be  seen  from  the  survey  of  the  bibliography 
of  the  subject  just  given,  the  conditions  of  the  viability  of  the 
pneumococcus  have  been  fairly  well  established  when  either 
sputum  or  other  fluids  containing  the  organism  are  dried  in  bulk 
and  exposed  to  diffuse  dayHght  or  the  direct  rays  of  the  sun. 
There  are  minor  inconsistencies  in  the  results  dependent  upon 
the  method  used,  the  sensitiveness  of  the  animal  employed  to 
determine  the  presence  of  Hving  pneumococci,  and  possibly  also 
upon  slight  variations  in  resistance  of  the  various  strains.  The 
identification  of  the  organism  was,  however,  so  far  as  is  reported 
in  many  of  the  studies,  entirely  dependent  upon  either  the 
morphology  of  the  bacteria  isolated  or  even  upon  the  death  of 
the  animal  without  any  microscopical  verification  of  the  presence 
of  a  septicemia.  As  it  has  been  shown  that  there  are  other 
capsulated  organisms  which  are  fatal  to  mice  if  given  in  suffi- 
ciently large  amoim.ts,  and  as  these  animals  and  also  rabbits 
frequently  die  after  the  injection  of  sputum  without  the  presence 
of  pneumococci  being  determinable  either  morphologically  or  by 
culture,  it  seemed  to  the  writer  that  a  few  experiments  might 
properly  be  devoted  to  a  repetition  of  the  studies  of  the  earlier 
Italian  and  German  workers  whose  papers  have  already  been 
considered.  The  experiments  of  Germano  in  mixing  cultures  of 
the  pneumococcus  or  sputum  with  sterile  dust  were  not  repeated 
for  they  are  ver^^  complete  as  they  stand  and  are  not  especially 
pertinent  to  the  question  in  hand. 

The  main  portion  of  the  writer's  studies  were  therefore  devoted 
to  the  investigation  of  the  question  of  fine  sprayed  particles  con- 
taining pneumococci  and  the  length  of  life  of  the  organisms  in 
this  spray.  This  ground  has  not  been  fully  covered  by  previous 
workers,  and  as  its  great  importance  in  the  transmissibility  of 
the  tubercle  bacillus  has  been  shown,  it  seemed  proper  to  extend 
our  knowledge  to  the  pneumococcus  although  it  has  generally 
been  assumed  that  that  organism  was  too  sensitive  to  desicca- 
tion to  live  very  long  in  fine  particles. 


Francis  Carter  Wood 


207 


I, EXPERIMENTS    ON    THE    VIABILITY    OF    THE    PNEUMOCOCCUS    IN 

LARGE    MASSES    OF    SPUTUM. 
Experiment  I. — The  following  tests  were  made  to  determine  the  viability 
of  the  pneumococcus  in  sputum  when  kept  moist  and  at  room  temperatures 
and  also  when  kept  at  o°  C. 

TABLE  I. 

TESTS    WITH    MOIST    SPUTUM. 


Day  of  Test. 

I 

5 

10 

15 

20 

30 

42 

60 

I.    Thick,    mucous    sputum 
from  3d  day  of  pneumonia 

22°C. 

o°C. 

+ 
+ 

+ 

+ 

0 

+ 

0 

+ 

0 

+ 

0 

+ 

0 

+ 

0 
0 

II.    Thick,      rusty      sputum 
from  3d  day  of  disease .... 

22°C. 

o°C. 

+ 
+ 

+ 

+ 

+ 
+ 

+ 

+ 

+ 
+ 

+ 
+ 

0 

+ 

0 

+ 

0 

+ 

0 
0 

0 
0 

III.    Thin,  fluid  sputum  from 
5th  day  of  disease 

20°  C. 
o°C. 

+ 
+ 

+ 

+ 

+ 
+ 

0 
0 

0 
0 

0 
0 

IV.    Thin,  yellowish  sputum 
after  crisis 

20°  C. 

o°C. 

+ 
+ 

0 
+ 

0 

+ 

0 

+ 

0 

+ 

0 

+ 

0 
0 

V.    Thick,  yellowish  sputum 
from  8th  day  of  disease . . . 

20°  C. 
o°C. 

+ 
+ 

+ 
+ 

+ 
+ 

+ 
+ 

0 

+ 

0 

+ 

0 
0 

0 
0 

The  positive  marks  mean  that  the  pneumococcus  was  either  isolated  from 
the  sputum  by  cultiore,  or,  especially  after  the  5th  day,  that  the  subcutaneous 
ijijection  of  from  one  fourth  to  one  fifth  of  a  cubic  centimeter  of  the  tmdiluted 
sputum  was  fatal  to  a  mouse.  No  result  was  considered  as  positive  unless 
capsulated.  Gram-positive  organisms  could  be  isolated  from  the  blood  of  the 
animal,  and  unless  the  coccus  fermented  inulin  after  plating  out  on  chest- 
serum  agar.  Occasionally  by  the  use  of  very  large  quantities  of  sputum  (0.5 
to  1.5  c.c.)  it  was  possible  to  kill  mice  up  to  fifty  days,  but  often  only  one  animal 
out  of  three  died,  showing  that  only  a  few  organisms  remained  alive. 

It  will  be  seen  from  the  table  that  the  life  of  the  pneumococcus 
in  fresh,  moist  sputum  at  room  temperatures  is  rarely  over  two 
weeks.  The  specimens  were  kept  in  the  dark  in  order  to  com- 
pare them  directly  with  those  at  o°  C,  which  were  of  necessity 
inclosed  in  a  cold-storage  box.  Two  specimens  kept  in  strong 
diffuse  daylight  lost  virulence  for  mice  in  less  than  five  days. 

The  rapid  death  of  the  organisms  in  sputum  as  compared  to 
chest-serum  is  possibly  due  to  the  bactericidal  action  of  the 
mucus  of  the  sputum. 

Experiment  II. — Tests  were  also  made  by  drying  sputum  in  Petri  dishes  at 
room  temperatures.  Some  of  the  specimens  were  kept  in  a  dark,  dry  spot, 
others  were  exposed  to  diffuse  daylight  in  a  room  facing  the  south,  others  were 
exposed  to  full  sunlight.  Fragments  of  the  dry  crust  of  sputum  were  then  re- 
moved, rubbed  up  in  sterile  bouillon,  and  inoculated  into  mice.    Other  specimens 


208 


Viability  of  the  Pnewmococcus  after  Drying 


were  finely  powdered  in  a  mortar  with  a  few  fragments  of  glass,  and  the  dust 
exposed  to  dajdight  or  direct  sunlight.     The  results  are  as  follows: 

TABLE  II. 

TESTS    WITH    DRIED    SPUTUM. 


Day  of  Test. 

I 

4 

8 

12 

20 

3° 

40 

60 

70 

80 

Sputum  kept  in  dark: 

No.     I.   Thin,  watery 

"     II.   Thick,  mucous — 

+ 
+ 

+ 
+ 

+ 
+ 

+ 
+ 

+ 
+ 

+ 
O 

+ 

0 

0 
0 

0 
0 

0 
0 

Sputum  exposed  to  diffuse 
light: 
No.      I.  Thin,  watery  .  .  . 
"      II.  Thick,     yellow, 

mucous 

"    III.  Thick  and  rusty . 

+ 

+ 
+ 

+ 

+ 

+ 

+ 

+ 
+ 

+ 

+ 
+ 

+ 

+ 
+ 

+ 

o 

+ 

0 
0 

+ 

0 

0 
0 

0 

0 

0 

0 

0 

0 

Sputum  dried  over  calcium 
chloride  in  daylight: 

No.  I.  Thin,  watery 

"  II.  Thick,  mucous  .  .  . 

+ 
+ 

+ 
+ 

+ 
+ 

+ 
+ 

+ 
+ 

+ 
+ 

+ 
+ 

+ 
+ 

0 

+ 

0 
0 

The  specimens  of  sputum  dried  over  calcium  chloride  retained  their  virulence 
for  mice  for  a  slightly  longer  period  than  those  exposed  to  the  air.  This  is 
probably  due  to  the  very  complete  and  prompt  drying  which  takes  place.  _  The 
specimens  exposed  to  the  air  never  dry  completely,  and  the  arnount  of  moisture 
retained  varies  from  day  to  day  in  accord  with  the  atmospheric  changes. 

TABLE  III. 

TESTS    WITH    DRIED    AND    PULVERIZED    SPUTUM. 


Hours  of  Test. 

I 

2 

4 

8 

12 

24 

36 

48 

Sputum  dried  and  exposed  to  sunlight : 
No       I          

+ 
+ 
+ 

+ 
0 

+ 

+ 
0 
0 

0 
0 
0 

0 
0 
0 

0 
0 
0 

0 
0 
0 

0 

"      II                 

0 

"    III 

0 

Sputum  finely  powdered  and  kept  in 
dark: 
No     I     

+ 
+ 

+ 
+ 

+ 
+ 

0 
0 

0 

+ 

0 
0 

0 
0 

0 
0 

0 
0 

0 
0 

0 
0 

0 
0 

0 
0 

0 
0 

0 

"      II      

0 

Exposed  to  diffuse  light : 

No     I                         

0 

"     II        

0 

Sputum  finely  powdered  and  exposed 
to  direct  sunlight: 
No.    I       

0 
0 

0 
0 

0 
0 

0 
0 

0 

0 

0 
0 

0 
0 

0 

"    II 

0 

Francis  Carter  Wood  209 

It  is  evident  from  the  table  that  the  exposure  of  the  pneumo- 
coccus  to  sunlight  results  in  the  prompt  death  of  the  organism. 
The  mere  powdering  of  the  sputum  also  destroys  the  pneumo- 
coccus,  a  phenomenon  probably  due  to  the  rapid  and  complete 
drying  which  takes  place.  The  action  of  even  diffuse  daylight  in 
hastening  the  death  of  the  organism  is  evident  from  the  table. 
Exposure  of  the  powder  to  sunlight  effects  an  even  more  rapid 
destruction,  there  being  probably  three  factors  in  the  process. 
One  is  the  formation  of  oxidizing  agents,  probably  hydrogen 
peroxide,  by  the  action  of  the  sun's  rays  upon  the  traces  of 
moisture  remaining  in  the  sputum, ^4  a  second  the  rapid  drying 
which  takes  place,  and  a  third,  the  destructive  action  of  the 
chemical  portion  of  the  sun's  rays. 

Experiment  III. — Sputum  was  spread  on  fragments  of  sterile  wood,  and  tin, 
and  on  woollen  and  cotton  cloth.  The  specimens  were  allowed  to  dry,  and 
were  kept  either  in  diffuse  daylight  or  sunlight.  The  life  of  the  organism  was 
about  the  same  on  wood  and  tin  as  on  glass.  On  cloth  several  tests  gave  a 
slightly  longer  life,  the  sputum  being  virulent  for  mice  after  sixty  days.  This 
is  explained  by  the  penetration  of  the  cloth  which  takes  place  when  soaked 
with  sputum,  the  fiber  of  the  cloth  protecting  the  organism  from  hght  and  the 
layer  of  sputum  formed  being  thicker  than  on  a  fiat  surface.  This  effect  was 
more  marked  in  those  fragments  exposed  to  sunlight,  one  piece  of  woollen 
cloth  being  virulent  to  mice  after  twelve  hours'  exposure,  about  six  hours  being 
given  on  two  successive  days  in  May.  The  death  of  the  bacteria  occurred  on 
two  hours'  further  exposure. 

II_ VIABILITY    OF    THE    PNEUMOCOCCUS    IN    FINE    SPRAYED 

PARTICLES. 

Technique. — In  order  to  spray  sputum  and  other  fluids  con- 
taining pathogenic  bacteria  and  to  collect  the  finer  particles,  it 
is  necessary  to  conduct  the  operation  in  an  air-tight  chamber, 
to  avoid  contamination  of  the  laboratory  and  infection  of  the 
operator. 

The  apparatus  employed  by  the  writer  was  modelled  upon  the  one  described 
by  Kirstein,3s  with  some  slight  modifications.  The  box  was  constructed  of 
seven-eighths  inch  white  wood  lumber  with  internal  measurements  of  38  cm.  in 
^gp^l^_  35  cm.  in  width,  and  152  cm.  in  length.     At  one  end  were  perforations 

34  Bie,  Mitth.  atis  Finsens  Med.  Lysinstitut,  1905,  Neuntes  Heft,  p.  5. 

35  Zeit.  f.  Hyg.,  1900,  xxxv,  145. 


210 


Viability  of  the  Pneumococcus  after  Drying 


for  the  insertion  of  the  tip  of  the  spraying  apparatus  and  apertures  to  per- 
mit of  the  escape  of  air  driven  into  the  chamber  while  spraying  the  sputum. 
In  order  to  prevent  direct  carrying  of  particles  the  full  length  of  the  chamber 
and  the  deposition  of  the  organisms  in  coarse  masses  upon  the  Petri  dishes  or 
other  substances  used  to  collect  the  spray,  two  bafHe  plates  were  placed  about 
the  middle  of  the  chamber,  22  cm.  apart.  These  plates  were  of  glass  and 
measured  28  by  35  cm.  They  were  held  in  place  by  narrow  strips  of  wood 
nailed  on  the  inner  side  of  the  box,  and  further  secured  by  putty  and  a  layer  of 
enamel  paint.  The  plate  nearer  the  spraying  apparatus  was  so  placed  that  its 
upper  portion^  was  in  contact  with  the  lid  of  the  box.  The  plate  farther  from 
the  spraying  apparatus  was  in  contact  with  the  floor  of  the  box,  leaving  a  space 
of  10  cm.  between  its  upper  edge  and  the  lid  (see  Fig.  i).  It  was  thus  im- 
possible for  particles  from  the  spray  to  pass  directly  from  one  end  of  the  box 
to  the  other.  The  coarser  masses  strike  the  first  plate  and  adhere  to  it.  Only 
the  finely  suspended  particles  pass  over  the  top  of  the  second  plate,  and  this 
only  when  a  current  of  air  is  drawn  through  the  apparatus  (see  Experiment 
IV). 


COMPARTMENT      -A- 


SPRAY  TU&t 


iHr 


COMPARTMENT    -B- 


fv--;r;.^-kSLv^sg-i:v-^vs.=!:--.s;ia*g.?M,--^^^s^^ 


Fig    I       Diagram  of  Box  for  Spraying  Pathogenic  Bacteria. 


In  order  to  collect  the  sprayed  particles,  three  apertures  were  made  at  the 
bottom  of  the  box,  two  of  which  were  circular,  measuring  13  cm.  in  diameter; 
the  third  was  rectangular  and  measured  26  by  14  era.  These  openings  had  a 
tin  collar  inserted  in  them  extending  about  10  cm.  below  the  bottom  of  the 
box.  These  collars  were  rendered  air-tight  by  white  lead.  Each  opening  was 
closed  during  the  experiment  by  placing  under  it  a  dish  some  3  or  4  cm.  larger 
in  diameter  than  the  collar,  the  dish  being  filled  with  i  :iooo  mercuric  chloride 
solution,  thus  making  a  water  seal.  The  dishes  were  held  in  position  by  the 
use  of  small  cupboard  buttons  which  could  be  swung  into  place  under  the  rim 
of  the  dish.  Before  the  spraying  was  commenced,  suitable  receptacles  for  catch- 
ing the  spray,  such  as  Petri  dishes,  either  dry  or  containing  culture  media,  or 
fragments  of  sterilized  cloth,  wood,  tin,  etc.,  were  placed  on  sraall  stands  which 
rested  on  the  bottom  of  the  dish.  For  the  smaller  apertures  in  the  first  com- 
partment these  stands  were  ordinary  drinking-glasses  which  were  inverted  and 
the  upper  end  surrounded  by  a  strip  of  half-inch  surgical  adhesive  plaster.  The 
Petri  dish  placed  on  this  adhered  quite  firmly,  and  there  was  no  danger  of  its 
falling  off  during  the  process  of  removal,  even  though  the  glass  was  consider- 


Francis  Carter  Wood  211 

ably  tipped.  In  the  larger  rectangular  opening  the  stand  was  made  of  half- 
inch  pine  board  with  four  nails  for  legs,  surrounded  by  a  collar  of  adhesive 
plaster.  This  collar  retained  the  plates  in  position  and  prevented  their  shifting 
during  insertion  or  removal  of  the  stand.  The  surface  of  the  Petri  plate  when 
inserted  was  approximately  level  with  the  bottom  of  the  spraying-box. 

In  order  to  render  the  inside  of  the  box  air-tight  and  waterproof,  the  corners 
were  filled  with  putty  and  the  inside  was  given  three  coats  of  thick  enamel 
bath-tub  paint.  The  lid  was  held  in  position  by  one-eighth  inch  steel  wires 
which  passed  from  a  turn  buckle  fastened  to  the  lid  on  one  side,  underneath 
the  box  and  over  a  wooden  brace  to  a  turn  buckle  on  the  opposite  side.  The 
turn  buckles  could  be  screwed  tight,  thus  holding  the  lid  firmly  in  position. 
In  order  to  make  a  suitable  seal,  the  upper  edge  of  the  box  was  smeared  with  a 
thick  layer  of  paste  sold  commercially  as  anti-phlogistine.  This  was  found  to 
be  better  than  putty  or  white  lead,  as  it  did  not  set,  but  remained  moist  and 
somewhat  pliable  for  a  period  of  nearly  two  months.  At  the  end  of  the  box 
farthest  from  the  spraying  apparatus  was  an  aperture  similar  to  those  in  the 
spraying  end  of  the  box.  A  suitable  opening  was  made  by  boring  a  hole  from 
the  outside  of  the  box,  about  22  mm.  in  diameter.  A  collar,  about  3  mm.  in 
width,  made  of  wood,  was  left  on  the  inside  of  this  opening.  Short  pieces  of 
brass  tubing  were  heated  over  a  Bunsen  flame,  smeared  with  rosin,  and  quickly 
inserted  in  the  holes,  into  which  they  fitted  snugly,  the  collar  of  wood  which 
was  left  insuring  a  firm  seat.  As  soon  as  the  rosin  cooled,  an  air-tight  joint 
was  obtained.  Four  of  these  apertures  were  made:  one  for  the  insertion  of  the 
spray  tube,  one  for  aspiration  of  the  air  current,  two  for  egress  of  the  air  forced 
in  by  the  presstire  apparatus.  The  spray  was  produced  by  a  long  glass  spray 
tube  which  was  inserted  through  one-  of  the  openings  and  tightly  packed  in 
position  with  absorbent  cotton.  After  the  spraying  was  completed,  these  tubes 
could  be  easily  sterilized  by  boiling  in  one  per  cent,  sodium  carbonate  solution. 
The  spraying  was  done  by  means  of  compressed  air,  the  pressures  used  varying 
from  five  pounds  or  less  to  the  square  inch  to  forty  pounds  to  the  square  inch. 
It  was  found  necessary  to  use  a  higher  pressure  in  the  case  of  thick,  mucous 
sputum  than  for  thin,  watery  sputum.  By  this  means  suitable  quantities  of 
thick  sputum,  in  many  instances  30  to  40  c.c,  could  be  atomized  during  the 
course  of  ten  minutes.  Before  beginning  the  experiments  the  box  was  tested 
by  closing  all  the  apertures  with  corks  and  inserting  a  water  manometer  in  one 
of  the  openings  and  forcing  in  air  through  another.  A  pressure  of  three  inches 
of  water  was  sustained  for  fifteen  minutes,  showing  that  the  box  was  air-tight 
for  this  pressure.  Higher  than  this  it  was  impossible  to  go  because  the  water- 
seals  would  have  been  forced  by  the  pressure.  As  it  was,  some  difficulty  was 
experienced  in  spraying  into  the  apparatus  unless  air  was  being  drawn  out  at 
the  same  time,  because  of  the  escape  of  bubbles  of  air  through  the  water-seals. 
It  would  be  advisable,  therefore,  to  modify  the  apparatus  and  make  the  seals 
somewhat  deeper;  possibly  15  cm.  would  be  better  than  the  10  cm.  used.  Air 
was  drawn  through  the  box  by  means  of  an  aspirating  bottle  graduated  in 
liters.  In  order  to  catch  any  bacteria  and  prevent  their  entry  into  the  aspirator,  a 
bottle  was  inserted  between  the  box  and  the  aspirator  containing  tightly  packed 
absorbent  cotton,  and  a  second  containing  sulphuric  acid  through  which  the  as- 
pirated air  was  forced  to  bubble.      The  rate  of  aspiration  could  be  measured 


212 


Viability  of  the  Pneumococcus  after  Drying 


by  timing  the  rate  of  outflow  from  the  bottle.  The  speed  at  which  the  par- 
ticles were  carried  from  the  chamber  nearer  the  spraying  apparatus,  which 
may  be  for  convenience  termed  A,  into  the  chamber  farther  from  the  spraying, 
called  B,  could  be  determined  as  follows.      (Fig.  2.) 

The  distance  between  the  two  baffle  plates  being  22  cm.,  their  height  28  cm., 
the  length  of  the  hypothenuse  would  be  about  36  cm.  The  diameter  of  the 
channel  is  then  approximately  6  cm.  The  cubic  contents  of  this  channel  from 
the  lower  aperture  of  the  flrst  baffle  plate  to  the  upper  aperture  of  the  second 
would  be  36  b)^  35  by  6  cm.  This  is  approximately  7560  c.cm.  If  this  amoiint 
of  air  is  aspirated  from  the  farther  end  of  the  box  in  one  minute,  the  velocity 
in  the  channel  will  be  6  mm.  per  second,  which  is  near  the  lowest  limit  of  air 
speed  which  will  move  very  fine  particles.  Air  speeds,  therefore,  were  used  in 
these  experiments  of  from  2  to  10  mm.  per  second. 


«=  — 22  cma 


-9r 


CO 


\^  / 


JL. 


Fig.  2.      Diagram  of  Dimensions  and  Course  of  Air  Current  in  Spraying  Box. 


Experiment  IV. — A  preliminary  experiment  made  with  bouillon  cultures  of 
B.  prodigiosus  showed  the  box  to  be  tight  and  that  no  bacteria  passed  the 
baffle  plates  unless  a  current  of  air  was  drawn  through  the  box. 

Sputum  was  then  obtained  from  a  case  of  acute  lobar  pneumonia  at  about  the 
third  day  of  the  disease.  Morphological  examination  showed  numerous  diplo- 
cocci  in  nearly  pure  culture.  They  were  positive  to  Gram,  but  no  capsules  were 
demonstrable.  The  sputum  was  plated  on  chest-serum  agar  and  diplococci 
isolated  which  were  Gram  positive,  had  well  marked  capsules,  fermented  inulin, 
and  killed  a  mouse  in  three  days,  capsulated  cocci  being  fotmd  in  the  heart's 
blood. 


Francis  Carter  Wood  213 

Ten  cubic  centimeters  of  this  thick  sptitum  were  sprayed  in  the  box,  using  40. 
pounds  air  pressure.  During  the  spraying  and  for  some  time  after,  a  slow  cur- 
rent of  air  was  drawn  through  the  apparatus  at  a  rate  of  about  0.2  mm.  per  second. 
Cover-slips  exposed  in  the  second  compartment  during  spraying  showed  nu- 
merous particles  derived  from  the  spray,  some  of  which  contained  pneumococci 
or  the  other  bacteria  of  the  sputum.  A  current  of  0.2  mm.  per  second  is 
therefore  capable  of  transporting  spray-carrying  bacteria  for  a  distance  of  at 
least  one  meter. 

After  the  spray  had  been  stopped,  covers  were  exposed  every  fifteen  minutes 
for  two  hours.  At  the  end  of  an  hour  most  of  the  particles  carrying  bacteria 
had  settled.  Covers  exposed  after  ninety  minutes  had  elapsed  showed  no 
bacteria,  only  very  small  particles  of  mucus  taking  a  blue  stain  with  gentian 
violet.  This  shows  that  the  bacteria  may  be  assumed  to  settle  38  cm.  in  from 
sixty  to  ninety  minutes. 

In  order  to  study  the  settling  of  the  particles  more  conveniently  and  to  de- 
termine the  length  of  time  for  which  sprayed  sputum  particles  can  remain  in 
suspension,  ten  cubic  centimeters  of  this  very  thick  sputum  were  sprayed  at  40 
pounds  pressure  into  a  tall  aspirating  jar  about  45  cm.  in  height,  the  air  contents 
of  which  had  been  cleansed  of  dust  by  aspiration  through  a  thick  cotton  plug. 
A  jar  was  used  for  this  preliminary  experiment  instead  of  the  box  just  de- 
scribed, because  of  the  ease  with  which  suspended  particles  could  be  rendered 
visible  by  a  strong  beam  of  light.  A  thick  fog  of  the  sprayed  particles  was 
produced  which  remained  suspended  for  sixteen  hours  and  could  be  rendered 
easily  visible  by  passing  a  beam  of  light  from  an  electric  arc.  At  the  end  of 
twenty-four  hours  only  a  few  fine  particles  could  be  seen  on  concentrating  the 
light  with  a  lens.  No  bacteria  were  deposited  on  cover-glasses  or  culture 
plates  after  the  jar  had  stood  for  two  hours. 

In  order  to  determine  whether  the  fine  spray  particles,  which  remained  a  long 
time  in  suspension  after  spraying  a  broth  culture,  contained  bacteria,  the  jar 
was  filled  with  spray  from  a  bouillon  culture  of  B.  prodigiosus,  and  after  stand- 
ing one  hour  the  plug  was  removed  from  the  upper  end  and  the  bottle  was 
reversed  and  allowed  to  rest  on  the  mouth  of  a  large  battery  jar.  Under  the 
mouth  was  placed  an  agar  covered  Petri  dish.  At  the  end  of  one  hour  the 
dish  was  removed,  covered,  and  allowed  to  remain  at  room  temperature  for 
several  days.     Abundant  growth  took  place. 

In  a  repetition  of  this  experiment  growth  was  obtained  by- 
allowing  the  fog  to  settle  on  plates  exposed  at  the  end  of  one 
hour  and  thirty  minutes,  but  no  growth  was  obtained  after  two 
hours,  nor  after  four  and  six  hours.  This  agrees  with  the  results 
obtained  by  Stern, ^^  who  states  that  ordinary  dust  particles  settle 
in  still  air  in  from  one  hour  and  a  half  to  three  hours,  and  can 
only  be  kept  afloat  by  air  currents  of  from  ten  to  thirty  millime- 
ters per  second.     Very  fine  particles  still  containing  bacteria  can 

3«  Zeit.  f.  Hyg.,  1889,  vii,  44. 


2 14  Yiability  of  the  Pneumococcij^  after  Drying 

be  transported  laterally  by  a  current  of  0.2  mm.  per  second,  and 
kept  afloat  by  a  current  of  from  0.3  to  0.4  mm.  per  second. ^^  The 
fog  made  evident  by  the  light  beam  after  a  period  of  from  five  to 
six  hours  is  probably  composed  of  dried  salts  and  albumin  or 
mucus  particles,  and  does  not  contain  bacteria. 

Air  currents  which  cause  the  movements  of  these  very  fine 
particles  have  been  shown  to  be  of  much  less  velocity  than  those 
which  occur  in  well  ventilated  rooms  where  the  motion  is  from 
I  to  2  mm.  per  second.  Air  at  ordinary  temperatures  does  not 
produce  a  perceptible  draught  until  its  velocity  reaches  10  cm.  per 
second.     In  unventilated  rooms  the  ciurent  is  less  than  0.6  mm. 

It  is  therefore  possible  for  spray  particles  containing  the 
pneumococcus  to  float  in  the  air  of  an  unventilated  room  for 
some  three  hours,  if  we  assume  the  rate  of  fall  as  determined  by 
the  experiments  to  be  at  least  30  cm.  per  hour  and  the  head  of 
the  patient  to  be  about  one  meter  from  the  floor.  With  air  cur- 
rents of  very  slight  intensity,  however,  the  finer  particles  may  be 
carried  for  considerable  distances.  Many  of  these  fine  droplets 
do  not  contain  bacteria,  so  that  the  practical  danger  from  a 
patient  with  pneumonia  is  less  than  appears  from  tests  under 
artificial  conditions.  The  coarse  particles  containing  many 
bacteria  fall  rapidly,  and  in  the  case  of  the  pneumococcus,  as  will 
be  seen  later,  many  of  the  suspended  organisms  lose  their  vitality 
in  the  course  of  one  or  at  most  two  hours. 

Experiment  V. — In  order  to  avoid  the  use  of  mixed  cultures  such  as  would 
be  obtained  from  sputum,  a  pleuritic  fluid  containing  enormous  numbers  of 
pneumococci  was  also  emploj^ed  in  the  studies.  This  fluid  was  obtained  by 
injecting  small  amounts  of  sputum  into  the  right  pleural  cavity  of  large  rabbits. 
The  injection  is  easily  made  by  passing  a  fine  needle  through  one  of  the  inter- 
costal spaces  on  the  lateral  aspect  of  the  thorax.  The  animals  usually  die  in 
two  to  three  days,  and  if  the  thorax  is  carefully  opened  from.  10  to  50  c.c.  of 
clear  or  slightly  bloody  fluid  can  be  obtained.  Usually  both  pleurae  and  the 
pericardial  sac  contained  fluid,  ^s 

Ten  cubic  centimeters  of  the  pleuritic  fluid  were  sprayed,  and  at  the  same  time 
sLxteen  liters  of  air  were  removed  at  such  a  rate  that  the  velocity  between  the 
baffle  plates  was  2  mm.  per  second.     The  control  plate  in  Chamber  A  was 

3'  Fliigge,  Zeit.  f.  Hyg.,  1897,  xxv,  193. 

3  8  This  method  of  obtaining  a  fluid  rich  in  pneumococci  was  suggested  to  me 
by  Dr.  A.  B.  Wadsworth.  The  organisms  remain  alive  in  the  serum  for  a  long 
time  at  0°  C. 


Francis  Carter  Wood  215 

removed  after  thirty  minutes  and  was  found  to  be  slightly  moist.  Bouillon 
was  poured  on  the  surface  and  rubbed  up  with  a  platinum  needle.  Loops  from 
the  bouillon  were  then  transferred  to  chest-serum  agar.  An  abundant  growth 
of  pneumococci  was  obtained.  Plates  exposed  in  Chamber  B  were  removed  at 
the  end  of  an  hour  and  a  fresh  set  inserted.  Cover  slips  which  had  been  ex- 
posed diiring  the  same  time  showed  numerous  capsulated  cocci.  Bouillon  was 
poured  over  one  plate,  rubbed  up  with  a  platinum  spatula,  and  injected  into  a 
mouse.  The  animal  died  from  pneumococcus  infection.  Other  plates  from 
Chamber  B  were  exposed  one,  two,  and  three  and  a  half  hours  to  diffuse  day- 
light. At  the  end  of  this  time  mice  were  inoculated  from  the  two-hour  plates, 
and  two  rabbits  3'  were  injected  in  the  ear  vein  with  an  emulsion  from  two 
three-and-a-half-hour  plates.  One  mouse  died  twenty  days  later,  but  no 
pneumococci  could  be  recovered.  The  other  lived  for  two  months.  The 
rabbits  did  not  die.  A  third  plate  after  three  and  a  half  hours  was  covered  with 
chest-serum  agar,  but  no  growth  was  obtained.  Another  plate  from  Compart- 
ment B  was  exposed  three  hours  to  sunlight  on  a  slightly  overcast  day.  Plates 
were  made  after  emulsifying  with  bouillon  and  three  mice  injected.  There  was 
no  growth  on  plates.  One  mouse  died  twelve  days  later,  but  no  pneumococci 
could  be  demonstrated.  The  others  did  not  die.  Another  plate  was  dried  over 
calcium  chloride  for  three  hours  in  diffuse  light.  Plate  cultures  and  animal  in- 
oculations were  negative. 

The  results  of  this  experiment  may  be  considered  as  showing 
that  spraying  a  thick  albuminous  fluid  containing  pneumococci 
and  allowing  the  fine  spray  to  dry  on  glass  is  fatal  to  the  organ- 
isms in  a  very  short  time.  If  drying  is  prevented  by  collecting 
the  particles  as  they  fall  on  moist  chest-serum  agar,  growth  will 
be  obtained  if  the  bacteria  have  not  been  in  suspension  over 
ninety  minutes.  A  fluid  of  the  type  used  corresponds  pretty 
closely  to  the  thin,  serous  sputum  of  certain  cases  of  pneumonia. 

Experiment  VI. — Twenty-five  cubic  centimeters  of  the  thick  sputum  used  in 
Experiment  IV  were  sprayed  in  the  box  during  ten  minutes,  using  an  air  current 
of  about  6  mm.  per  second.  Control  plates  from  Compartment  A  were  positive. 
Plates  from  Compartment  B  removed  immediately  were  negative  to  mice,  and 
cultures  gave  only  staphylococci.  Cover-glasses  showed  numerous  drops 
varying  from  three  to  fifteen  micra  in  diameter.  The  larger  drops  frequently 
contained  two  or  three  diplococci. 

One  hoiir  after  spraying,  cover-glasses  were  placed  in  Compartment  B  and 
allowed  to  remain  fifteen  hours.  These  showed  only  a  few  bacteria,  less  than 
one  per  square  centimeter.  There  were,  however,  many  masses  of  mucus 
which  had  fallen  on  the  slide,  most  of  which  did  not  contain  bacteria.  No  free 
organisms  were  found,  all  were  surrounded  by  more  or  less  mucus. 

3  9  It  was  thought,  inasmuch  as  the  pneumococci  used  had  been  adapted  to 
rabbits,  that  these  animals  might  be  more  susceptible  to  infection  than  mice. 


216  Viability  of  the  Pneumococcus  after  Dryinuj 

Chest-serum-agar  plates,  exposed  in  Compartment  B  for  a  period  beginning 
one  hour  after  the  spraying  was  finished,  gave  abundant  growth  of  staphylo- 
cocci, but  mice  injected  with  an  emulsion  showed  no  pneumococci.  Some 
plates  remained  in  Compartment  B  for  a  number  of  days,  but  no  pneumococci 
could  be  demonstrated.  Dry  plates  remained  ten  days,  and  when  covered  with 
agar  gave  numerous  colonies  of  Staphylococcus  pyogenes  aureus.  Mice  which 
were  injected  died,  but  no  pneumococci  could  be  isolated.  Plates  from  this 
spraying  were  kept  in  the  dark  and  their  contents  injected  into  mice  at  various 
intervals.     Some  of  the  animals  died,  but  no  pneumococci  could  be  obtained. 

A  plate  d:fted  over  calcium  chloride  for  fifteen  hours  in  the  dark  gave  no 
pneumococci,  but  only  staphylococci. 

The  results  of  the  experiment  show  the  rapidity  with  which 
the  pneumococcus  dies  when  sprayed  in  fine  particles  and  allowed 
to  dry.  The  drying  seems  to  be  an  important  factor,  for  if,  as 
is  shown  in  Experiment  V,  the  bacteria  are  caught  on  moist 
media,  a  growth  will  be  obtained.  As  a  rule  also  we  must  assume 
that  there  are  other  factors  at  work,  for  the  viability  of  the  organ- 
isms after  spraying  sputum  is  certainly  less  than  that  after 
spraying  rabbit  chest-serum.  The  action  of  the  mucus  must  be 
considered  and  possibly  also  the  osmotic  relations  of  the  organism 
to  the  sputum  may  affect  the  pneumococcus  unfavorably.  It  is 
also  not  impossible  that  a  considerable  proportion  of  the  pneumo- 
cocci in  sputum  from  the  later  stages  of  the  disease  are  not 
viable. 

Experiment  VII. — Forty  cubic  centimeters  of  fresh,  thin,  serous  sputum, 
from  the  sixth  day  of  the  disease,  one  loop  of  which  was  capable  of  killing  a 
mouse  in  forty-eight  hours,  were  sprayed  for  one  hour  with  an  air  current  of  lo 
mm.  per  second.  The  control  plate  from  Chamber  A  killed  a  mouse  in  forty- 
eight  hours.  Pneumococci  were  isolated  which  were  positive  to  Gram,  were 
capsulated,  and  fermented  inulin.  The  plate  was  dry  when  it  was  removed 
from  the  compartment. 

Plates  from  Chamber  B,  removed  at  the  end  of  spraying  and  found  to  be  dry, 
were  washed  with  bouillon  and  the  washings  injected  into  two  mice.  One  died, 
but  no  pneumococci  were  found;  the  other  lived  a  month.  Washings  from 
these  plates  were  sown  on  the  surface  of  chest-serum  agar  in  order  to  avoid  the 
inhibiting  action  of  the  anaerobic  conditions  which  exist  under  a  layer  of  agar. 
Staphylococci  and  other  unidentified  organisms  were  obtained,  but  no  pneumo- 
cocci. 

Plates  dried  over  calcium  chloride  were  also  negative  as  re- 
gards pneumococci.  These  experiments  were  repeated  with 
sputa  from  different  cases  and  at  different  times  of  the  disease, 
but  the  results  were  practically  the  same. 


Francis  Carter  Wood  217 

It  is  evident  that  finely  sprayed  sputtim  contains  no  viable 
pneiunococci  after  drying  on  glass  for  one  hour.  The  positive 
results  occasionally  obtained  from  the  Control  plates  in  com- 
partment A  may  be  explained  by  the  thick  layer  of  sputum 
which  is  deposited  and  prevents  complete  desiccation.  The 
results  obtained  by  using  thin,  serous  sputum  do  not  vary 
from  those  obtained  when  thick,  mucous  sputum  was  sprayed, 
although  differences  appear  when  the  sputa  are  dried  in  bulk. 

Experiment  VIII. — The  technique  was  varied  slightly  so  as  to  transfer  large 
numbers  of  the  organisms  and  thus  to  keep  them  moist.  About  loo  c.c.  of 
pleuritic  fluid  were  sprayed,  with  an  air  current  of  lo  mm.  per  second.  Fifteen 
minutes  after  spraying  was  completed  the  plates  in  Compartment  B  were  re- 
moved. They  were  still  moist.  A  mouse  injected  with  an  emulsion  of  the 
deposit  died  with  pneumococci  in  the  heart's  blood.  A  plate  was  exposed  to 
sunshine  for  twenty  minutes,  during  which  time  it  dried.  A  mouse  injected  did 
not  die.  A  plate  of  the  same  series  was  dried  over  calcium  chloride  for  thirty 
minutes.  One-half  was  injected  into  a  mouse  which  died  with  pneumococcus 
sepsis.  The  plate  was  dried  thirty  minutes  more;  mouse  died  with  pneumo- 
cocci in  the  heart's  blood.  A  plate  from  the  same  series  was  dried  two  hours 
over  calcium  chloride;  a  mouse  injected  did  not  die.  A  broth  culture  was 
made  from  this  plate  and  showed  pneumococci  which  killed  a  mouse  (see  results 
obtained  by  Ottolenghi).  Another  plate  was  dried  for  three  hours  and  a  mouse 
injected  and  a  broth  culture  made.  Broth  was  negative  and  the  mouse  re- 
mained alive.  Plates  dried  in  air  for  one  hour  were  positive;  for  one  and  a  half 
and  two  hours,  negative. 

It  is  possible  that  when  only  a  small  quantity  of  fluid  is  sprayed  there  are  not 
enough  virulent  pneumococci  left  after  drying  to  kill  the  experimental  animal. 
A  certain  minimum  dose  seems  necessary  to  kill  even  as  susceptible  an  animal 
as  a  white  mouse.  A  larger  quantity  of  fluid  was  therefore  sprayed  in  this  test, 
and  as  shown  by  the  slightly  longer  life  of  the  pneumococci  as  compared  to 
Experiments  V,  VI,  and  VII,  the  quantity  exerts  some  influence.  The  condi- 
tions approach  those  which  occur  in  drying  sputum  in  bulk  (see  Table  II)  where 
the  life  of  the  organism  is  considerably  prolonged. 

During  this  test  fragments  of  sterilized  woollen  and  cotton  cloth,  tin,  and 
wood  were  exposed  in  Compartment  B.  They  were  removed,  allowed  to  dry  in 
the  air  for  thirty  minutes,  and  scrapings  from  the  surface  tested.  The  organ- 
isms on  the  tin  and  wood  were  dead,  those  on  the  cloth  were  alive,  but  died  on 
drying  for  thirty  minutes  longer. 

Experiment  IX. — A  number  of  observers  have  thought  that  the  pneumo- 
coccus in  sputum  is  rapidly  destroyed  by  the  bactericidal  action  of  the  mucus 
of  the  sputum.  Such  action  has  been  shown  to  take  place  with  nasal  and  uterine 
mucus  and  pure  mucin. ■lo 

*o  Wurtz  and  Lemoyez,  Compt.  rend,  de  la  Soc.  de  Biol.,  1894;  Arloing,  Jour, 
de  phys.  et  de  path,  g^n.,  1902,  iv,  291  (Bibliography). 


218 


Viability  of  the  Pneitmococcus  after  Drying 


(a)  This  was  tested  by  keeping  a  thick,  mucous  sputum  at  o°  C,  as  recorded 
in  Experiment  I.  As  the  pneumococcus  dies  on  cvdture  media  or  in  rabbit 
serum  in  a  few  days  unless  kept  at  o°  C,  it  was  thought  that  a  better  differentia- 
tion could  be  obtained  by  working  at  the  lower  temperature  and  in  the  dark. 
When  first  collected  the  specimens  killed  mice  in  doses  of  a  few  cubic  milli- 
meters in  forty-eight  hours.  After  fifteen  days  at  o°  C,  a  much  larger  amount 
of  sputum  was  required  to  kill  a  mouse  of  about  the  same  size  as  that  used 
during  the  first  experiment.  At  the  end  of  six  weeks  mice  often  could  be  killed 
only  by  doses  of  a  cubic  centimeter  of  pure  sputum,  while  one  specimen  was  no 
longer  viruttnt  after  twenty  days.  Evidently  a  large  number  of  the  pneumo- 
cocci  die  in  two  weeks  when  kept  in  moist  sputum. 

As  it  is  well  established  that  the  pneumococcus  remains  alive  for  a  long 
period  when  kept  in  serum  mixtiires  at  o°  C,  a  combination  of  this  fluid  with 
sputum  should  retain  its  virulence  as  long  as  pure  sputum  unless  some  bacteri- 
cidal agent  is  present  in  the  sputum.  Such  a  mixture  was  therefore  made  and 
kept  in  Petri  dishes.  The  sputa  used  were  the  same  as  in  Table  I.  The  results 
were  as  follows: 

TABLE  IV. 

TESTS    WITH    SPUTUM-CHEST-SERUM    MIXTURES    KEPT    AT    0°    C. 


Day  of  Test. 

I 

5 

lO 

15 

20 

30 

42 

6o 

Sputum  No.  I. : 

+  serum  containing  pneumococci . 

4- 

+ 

+ 

+ 

+ 

+ 

+ 

o 

Sputum  No.  II.: 

+  serum  containing  pneumococci. 

+ 

+ 

+ 

+ 

+ 

+ 

O 

o 

Sputum  No.  III.: 

-I-  serum  containing  pneumococci. 

+ 

+ 

+ 

+ 

+ 

-1- 

+ 

o 

Sputum  No.  IV. : 

-I- serum  containing  pneumococci. 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

o 

Sputum  No.  V. : 

-1-  serum  containing  pneumococci. 

+ 

+ 

+ 

+ 

+ 

O 

O 

o 

Serum  alone 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

+ 

The  table  shows  that  the  senim-sputum  mixtures  do  not  retain 
their  virulence  for  mice  much  longer  than  the  original  unmixed 
sputum  as  given  in  Table  I.  In  two  cases,  however,  that  of 
sputum  No.  Ill  and  No.  IV,  virulent  pneumococci  were  still 
present  at  the  end  of  six  weeks,  while  the  pure  sputum  was  non- 
virulent  after  three  weeks'  preservation.  This  difference  is 
possibly  due  to  the  fact  that  but  little  mucus  was  present  in  the 
sputum.     The  practical  importance  of  these  findings  is  that  the 


Francis  Carter  Wood 


219 


thin,  serous  sputa  are  likely  to  retain  their  infectious  qualities 
somewhat  longer  than  the  thick,  mucous  specimens,  and  as  the 
thin  sputa  are  most  easily  sprayed  during  coughing,  special  care 
should  be  taken  to  avoid  contact  infections. 

In  order  to  determine  the  action  of  the  mucus  during  spraying 
and  after  drying  of  the  spray  particles,  the  following  experiment 
was  planned. 

(b)  Specimens  of  sputa  Nos.  VI  and  VII  were  mixed  with  an  equal  quantity 
of  rabbit  chest-serum  rich  in  pneumococci.  Mice  injected  with  the  mixture 
died  promptly  of  pneumococcus  infection.  The  specimens  were  kept  on  ice  in 
the  dark,  and  in  diffuse  daylight  at  room  temperature.  The  results  were  as 
follows : 

TABLE  V. 

TESTS    WITH    SPRAYED    SPUTUM-CHEST-SERUM    MIXTURE. 


Sprayed  after 

2 
days. 

A 

days. 

8 
days. 

IS 
days. 

20 
days. 

Sputum  VI.: 

+  serum     on    ice    in 

dark. 

Thin,  serous  sputum. 

Plates  removed  from  Comp. 
B.  immediately 

Dried  in  air  for  30  min 

"  sunlight  for  30  min. 

+ 

+ 
0 

0 
0 

0 
0 

+ 

+ 

Sputum  VI. : 

+  serum  in  dark  at 
room  temp. 

Same  conditions  as  above. 

+ 
0 
0 

0 
0 
0 

-_ 

— 

I 

Sputum  VII.; 
+  serum  on  ice. 
Thick  mucous    spu- 
tum. 

Same  conditions  as  above. 

+ 
0 
0 

+ 
0 
0 

0 
0 
0 

- 

- 

Sputum  VII.: 

+  serum     at    room 
temp.  (i8°-22°  C). 

Same  conditions  as  above. 

0 

0 
0 

0 
0 
0 

0 
0 
0 

— 

— 

Sputum  VI.: 

Without   admixture 
on  ice. 

Same  conditions  as  above. 

+ 
0 
0 

-1- 
0 
0 

+ 
0 
0 

0 

- 

Sputum  VII.: 

Without   admixture 
in   dark   at   room 
temp. 

Same  conditions  as  above. 

+ 
0 
0 

0 
0 
0 

0 
0 
0 

Chest-serum  on  ice .... 

+ 

-t- 

+ 

+ 

+ 

Chest-serum  in  light. .  . 

+ 

4- 

+ 

0 

220  Yiahility  of  the  Pneumococcvs  after  Drying 

The  results  of  the  experiments  show  that  pneumococci  die  off 
in  mucous  sputimi  more  rapidly  than  they  do  in  a  serum  mixture 
and  that  this  action  is  probably  due  to  the  mucus  present.  The 
pure  serum  used  in  this  test  preserved  its  virulence  for  weeks 
when  kept  on  ice,  and  for  eight  days  in  diffuse  light, 

ExPERiMEXT  X. — In  order  to  determine  whether  the  rapid  death  of  the 
sprayed  organisms  is  due  to  the  drying  which  takes  place  while  they  are  sus- 
pended in  the  air  or  after  they  are  deposited  on  the  glass  plates  or  other  dry 
substances  used  to  collect  them,  the  following  variation  was  made  in  the  test. 

Thirty  cubic  centimeters  of  pleuritic  fluid  were  sprayed,  using  an  air  current 
of  lo  mm.  per  second.  The  contents  of  the  control  plates  removed  at  the  end 
of  the  spraying  killed  mice  in  two  days  and  gave  an  abundant  growth  on  serum- 
agar.  Plates  of  serum-agar  were  inserted  in  Compartment  B  at  the  completion 
of  the  spraying  and  the  air  current  was  continued  for  thirty  minutes.  These 
plates  were  removed  in  thirt}'  minutes  and  a  second  set  of  serum-agar  plates 
was  substituted.  These  were  also  removed  in  thirty  minutes  and  a  fresh  set 
substituted. 

On  the  first  group  of  plates  there  was  an  abundant  growth  of  pneumococci 
which  killed  mice — i.  e.,  had  lost  none  of  their  virulence.  The  second  set  of 
plates  showed  about  twenty  colonies  each.  These  were,  of  course,  derived  from 
bacteria  which  had  been  in  suspension  for  at  least  thirty  minutes.  The  third 
set  inserted  at  the  end  of  an  hour  after  the  spraying  had  ceased  and  allowed  to 
remain  for  three  and  a  half  hours,  showed  one  or  two  colonies  of  pneumococci. 
Cover-glasses  inserted  at  the  same  time  showed  no  demonstrable  pneumococci 
after  a  long  search  and  only  small  masses  of  deposited  spray.  It  is  evident  that 
practically  all  the  bacteria  had  settled  out  from  a  height  of  38  cm.  in  an  hour's 
time,  and  that  those  in  suspension  for  that  time  were  still  alive,  probably  owing 
to  their  being  protected  from  complete  desiccation  by  the  inspissated  serum 
surrounding  them.  In  order  to  extend  the  time  dtuing  which  the  organisms 
could  be  suspended  in  the  air,  a  sputum-chest-serum  mixture  was  sprayed  into 
a  tall  aspirating  jar  some  45  cm.  in  height.  As  the  rate  of  fall  in  still  air  of  fime 
particles  containing  pnetmaococci  is  about  40  cm.  per  hour,  the  jar  was  inverted 
every  fifteen  minutes  for  two  hovu"s,  diiring  which  time  it  was  exposed  to  diffuse 
light.  It  was  then  fixed  mouth  do-RTiward  over  a  Petri  dish  containing  chest- 
serum  agar  and  was  left  for  six  hours  in  the  dark.  Numerous  colonies  of  Staphy- 
lococcus pyogenes  aiu"eus  developed,  but  none  of  the  pneumococcus. 

As  shown  above,  the  organism  is  alive  after  an  hour's  suspen- 
sion. A  second  test  showed  that  only  a  few  pneumococci  survive 
for  ninety  minutes  when  suspended  in  a  fine  spray  in  diffuse  light. 
Such  a  fact  is  of  the  greatest  importance  from  a  point  of  view  of 
the  hygiene  of  those  in  close  contact  with  persons  suffering  with 
pneumonic  infections.  It  demonstrates  the  necessity  of  an 
abundant  air  supply  to  dilute  the  cloud  of  organisms  which  sur- 


Francis  Carter  Wood  221 

rotind  a  patient  with  a  severe  cough.  A  repetition  of  the  same 
test  allowing  the  jar  to  stand  in  direct  sunlight  for  fifteen  and 
thirty  minutes,  and  then  removing  it  to  a  dark  room  to  permit 
the  organisms  to  settle,  show^ed  in  a  very  striking  manner  the 
value  of  sunlight  as  a  disinfectant.  Only  a  few  colonies  of  the 
pneumococcus  were  obtained  after  fifteen  minutes,  and  none  at 
the  end  of  half  an  hour, 

SUMMARY    AND    CONCLUSIONS. 

I.  In  moist  sputum  kept  in  the  dark  at  room  temperatures 
the  average  Hfe  of  the  pneumococcus  is  eleven  days,  though  con- 
siderable variations  ma}^  be  noted  in  different  specimens  of 
sputum. 

In  the  same  sputum  kept  at  o°  C.  the  average  life  of  the  organ- 
ism is  thirty-five  days. 

In  sputum  kept  at  room  temperature  and  in  a  strong  light  the 
pneumococcus  lives  less  than  five  days. 

II.  In  dried  sputum  (a)  in  the  dark  the  pneumococcus  lives 
on  an  average  thirty-five  days ;  (6)  in  diffuse  light,  thirty  days ; 
(c)  in  sunHght,  less  than  four  hours. 

III.  In  powdered  sputum  even  when  kept  in  the  dark  the 
death  of  the  pneumococcus  takes  place  in  from  one  to  four  hours. 
When  exposed  to  sunlight  death  occurs  within  an  hour. 

IV.  No  important  differences  were  noted  in  the  life  of  the 
pneumococcus  when  dried  on  glass,  tin,  or  wood.  On. cloth  the 
life  was  usually  slightly  longer  than  on  non-absorbing  surfaces. 

V.  Sprayed  sputum  particles  remain  in  suspension  for  twenty- 
four  hours,  but  all  masses  of  a  size  sufficient  to  contain  bacteria 
settle  at  a  rate  of  about  40  cm.  per  hour. 

VI.  When  sputum  containing  pneumococci  is  sprayed  the 
organisms  rarely  sur\Kve  for  more  than  an  hour,  and  often  die  in 
less  time.  The  substance  upon  which  the  particles  fall  makes 
but  little  difference  in  the  life  of  the  organism.  On  cloth  a  slight 
prolongation  is  occasionally  noted,  due  perhaps  to  the  slow 
drying. 

VII.  The  mucus  of  the  sputum  exerts  a  destructive  action 
on  the  pneumococcus. 


222  Viability  of  the  Pneumococcus  after  Drying 

VIII.  Exposure  of  bacterial  spray  to  sunlight  while  in  sus- 
pension results  in  the  destruction  of  the  pneumococcus  within 
half  an  hour. 

IX.  The  conclusions  of  practical  importance  which  can  be 
drawn  from  the  facts  given  in  this  paper  are  as  follows : — 

A.  The  life  of  the  pneumococcus  in  moist  sputum  is  of  con- 
siderable duration,  the  average  period  being  less  than  two  weeks 
unless  the  material  is  exposed  to  direct  sunlight.  But  as  such 
sputum  does  not  give  off  bacteria  even  when  exposed  to  strong 
currents  of  air,  it  may  be  considered  as  innocuous  except  to 
persons  handling  clothes,  bedding,  etc.,  which  have  recently 
been  contaminated.  Under  ordinary  conditions,  however,  this 
sputum  dries  in  the  course  of  a  few  hours  or  days.  The  dried 
masses  retain  their  virulence  for  a  long  time,  and  if  deposited  on 
the  floor  or  on  the  bedding  of  the  patient  may  be  powdered 
mechanically,  and  sweeping,  dusting,  or  brushing  the  con- 
taminated articles  will  distribute  pneumococci  in  the  air.  Fortu- 
nately, however,  the  organisms  in  the  sputum  do  not  remain  long 
in  suspension  and  die  off  rapidly  under  the  action  of  Hght  and 
desiccation.  In  sunlight  or  diffuse  dayhght  the  bacteria  in  such 
powder  die  within  an  hour,  and  in  about  four  hours  if  kept  in  the 
dark.  The  danger  of  infection  from  powdered  sputum  may, 
therefore,  be  avoided  by  ample  illumination  and  ventilation 
of  the  sick-room  in  order  to  destroy  or  dilute  the  bacteria,  and 
by  the  avoidance  of  dry  sweeping  or  dusting.  Articles  which 
may  be  contaminated  and  which  cannot  be  cleaned  by  cloths 
dampened  in  a  suitable  disinfectant  should  be  removed  from  the 
patient's  vicinity. 

B.  When  a  person  suffering  from  a  pneumococcus  infection 
coughs,  sneezes,  expectorates,  or  talks,  particles  of  sputum  or 
saliva  are  expelled  from  the  mouth  which  may  contain  virulent 
pneumococci.  Such  particles  remain  suspended  in  the  air  for  a 
number  of  hours  if  the  ventilation  of  the  room  is  good.  They 
may  be  inhaled  by  persons  in  the  vicinity  of  the  patient,  or  they 
may  be  deposited  upon  various  articles  in  the  room.  Whether 
suspended  in  the  air  or  dried  on  surrounding  objects,  the  writer's 
studies  show  that  they  become  harmless  in  a  very  short  time, 


Francis  Carter  Wood  223 

about  an  hour  and  a  half  being  the  extreme  limit,  while  many 
of  the  pneumococci  in  the  spray  perish  in  a  few  minutes,  espe- 
cially if  exposed  to  strong  light. 

In  the  light  of  these  experiments  the  risk  of  infection  from 
the  pneumococcus  is  largely  confined  to  those  in  direct  contact 
with  the  person  whose  excreta  contain  the  organism. 

The  writer  wishes  to  acknowledge  his  obligations  to  Prof.  T. 
Mitchell  Prudden  for  many  helpful  suggestions  made  during  the 
course  of  this  study. 


A  NOTE  UPON  THE  GROWTH  OF  PNEUMOCOCCI  AND 
STREPTOCOCCI  IN  BLOOD  SERUM. 

By  WARFIELD  T.  LONGCOPE,  M.D. 
Director  "of  the  Ayer  Clinical  Laboratory  of  the  Pennsylvania  Hospital. 

During  the  investigations  described  in  another  paper  defibri- 
nated  blood  and  blood  serum  were  frequently  used,  either  alone 
or  mixed  with  agar,  as  media  upon  which  to  grow  pneumococci 
and  streptococci.  It  was  immediately  noted  that  the  organisms 
grew  very  differently  in  serum  obtained  from  different  individuals. 
The  variations  were  so  marked  and  the  results  obtained  so 
striking  that  it  was  decided  to  study  the  matter  more  carefully. 
To  this  end  the  growth  of  pneumococci  and  streptococci  was 
studied  in  the  blood  serum  of  thirty-two  individuals.  Besides 
the  human  sera,  horse's  serum,  calf's  serum,  and  rabbit's  serum 
were  used. 

In  the  researches  conducted  by  Besangon  and  Griffon,^  Huber,2 
Neufeld,3  and  Wadsworth  ^  upon  the  agglutination  of  pneu- 
mococci by  the  serum  of  pneuraonia  patients  and  of  immunized 
animals,  certain  changes  besides  the  actual  agglutination  were 
noted  in  the  bacteria  and  serum.  Huber  in  particular  calls 
attention  to  the  heavy  precipitate  formed  by  growing  pneu- 
mococci in  pneumonic  serum,  a  phenomenon  which  did  not 
occur  in  the  serum  from  normal  individuals,  when  used  as  a 
culture  medium  for  pneumococci.  Neufeld  describes  swelling 
of  the  capsules  of  the  pneumococci  when  treated  with  the  sertim 
from  cases  of  pneumonia  or  from  immunized  animals.  Wads- 
worth  has  confirmed  these  observations.  Recently  Rosenow  ^ 
has  called  attention  to  another  very  interesting  result  of  the 

>  Annal.  de  I'lnst.  Pasteur,  1900,  xiv,  449. 

2  Cent.  f.  inn.  Med.,  1902,  417. 

3  Zeit.  f.  Hygiene  u.  Infectionskrank.,  1902,  xl,  54. 
*  Journal  of  Med.  Research,  1903,  x,  228. 

•5  Journal  of  Infectious  Diseases,  1904,  ii,  280. 

224 


y 

/' 

Warfiekl  T.  Longcope  225 

growth  of  pneumococci  m  pneumonic  serum.  After  two  or 
three  days'  growth  acid  is  formed  in  the  serum.  This  does  not 
occur  when  the  senmi  from  normal  individuals  is  used.  Scarlet- 
fever  serum,  however,  did  yield  acid.  The  rich  precipitate 
which  forms  in  the  serum  after  the  growth  of  pneimiococci  is  due, 
at  least  in  part,  as  Rosenow  thinks,  to  the  action  of  the  acid  upon 
the  serum,  and  is  not  entirely  formed,  as  was  thought  by  Huber 
and  Wadsworth,  by  masses  of  agglutinated  pneimiococci. 

The  pneumococci  in  the  present  study  were  obtained  both 
from  the  saliva  of  normal  individuals  and  from  the  consolidated 
lungs  in  cases  of  lobar  pneumonia.  The  streptococci  were 
procured  from  the  saliva  of  normal  individuals  and  from  patho- 
logical conditions. 

The  serum  was  obtained  from  persons  suffering  from  various 
diseases.  In  all  there  were  fifteen  cases  of  pneumonia,  five  of 
chronic  nephritis  and  uremia,  two  of  general  streptococcus 
infection,  one  case  of  gonorrhoeal  endocarditis  (gonococcus  re- 
covered in  blood  culture),  three  cases  of  tuberculosis,  one  case 
of  acute  articular  rheumatism,  four  cases  in  which  the  diagnosis 
was  not  absolutely  certain,  and  one  normal  individual.  To 
collect  the  serum,  about  20  c.c.  of  blood  were  drawn  from  the 
arm  vein  and  allowed  to  clot  in  a  cool  place ;  after  from  twenty- 
four  to  forty-eight  hours  the  serum  was  drawn  off.  The  proper- 
ties of  the  serum,  so  far  as  the  growth  of  the  pneumococci  and 
streptococci  was  concerned,  did  not  alter  with  age.  The  same 
reactions  were  seen  in  the  serum  twenty-four  or  forty-eight  hours 
and  several  weeks  after  the  blood  was  drawn.  From  2  to  5  c.c. 
of  serum  were  used  as  a  culture  medium. 

In  the  normal  serum  the  pneumococci  produced  a  slight  cloud, 
the  streptococci  a  granular  sediment.  The  serum  remained 
alkaline.  In  the  serum  from  the  cases  of  pneimionia  the  growth 
of  the  pneumococcus  was  extremely  interesting.  After  twenty- 
four  hours  there  was  a  diffuse  cloud,  often  with  a  heavy,  whitish 
precipitate,  and  the  fluid  appeared  a  little  thick.  The  reaction 
varied,  with  litmus  as  an  indicator,  from  neutral  to  well-marked 
acidity.  In  hanging-drop  preparations  small  clumps  of  highly 
refractile  bodies  were  seen.     They  were  two  or  three  times  the 


226  Growth  of  Pneumococci  and  k....'eptococci 

size  of  pneumococci  but  had  much  the  same  morphology  and 
appeared  as  pneumococci  with  greatly  swollen  capsules.  Besides 
these  swollen  clumps  there  were  many  single  cocci  which  were 
not  swollen  and  many  minute  granules,  the  morphology  of  which 
could  not  be  definitely  determined.  Preparations  stained  with 
the  usual  dyes  showed  well  preserved  pneumococci  with  capsules. 
The  clumps  of  swollen  refractile  bodies  stained  poorly  and  only 
occasionally  could  they  be  identified  as  bacteria. 

With  increased  growth  the  cloud  and  thickening  progressed 
until  after  from  six  to  eight  days  the  fluid  was  opaque,  white,  of 
almost  semi-solid  consistency,  and  looked  much  like  highly 
albuminous  urine  after  treatment  with  heat  and  acetic  acid. 
During  the  growth  the  acidity  steadily  increased.  At  the  end  of 
from  six  to  eight  days'  growth,  it  usually  required  0.3  to  0.7  c.c. 
of  Y^j-  N.  sodium  hydroxide  to  neutralize  i  c.c.  of  the  serum,  when 
litmus  was  used  as  an  indicator.  With  continued  growth  the 
masses  of  swollen  refractile  bodies  increased  greatly  in  size  and 
numbers ;  the  remainder  of  the  cloud  seemed  to  be  made  up  of 
fine  granules.  With  the  various  dyes  only  a  few  pneumococci 
took  the  stain.  The  streptococci  grew  in  an  entirely  different 
manner.  They  usually  produced  no  cloud  but  a  fine  granular 
sediment,  often  grew  very  poorly,  and  never  gave  rise  to  an  acid 
reaction. 

The  heavy  clouding  formation  of  precipitate,  thickening,  and 
acid  production  took  place  in  the  serum  from  all  but  four  of  the 
cases  of  pneumonia.  In  two  of  these  instances  the  blood  was 
drawn  after  the  crisis ;  and  in  the  others  on  the  day  of  the  crisis 
and  during  a  post-pneumonic  empyema  respectively.  Table  I 
gives  the  days  upon  which  the  blood  was  drawn. 

The  reaction  just  described  was  not  found  to  be  specific  for 
pneiunonic  sera.  Practically  the  same  alterations  were  observed 
when  pneumococci  were  grown  in  the  sera  from  two  cases  of 
general  streptococcus  infection,  from  a  case  of  gonococcus 
endocarditis,  and  from  a  case  of  acute  articular  rheumatism. 
Slight  clouding  and  moderate  quantities  of  acid  were  formed 
with  the  sera  from  the  five  cases  of  chronic  nephritis  and  uraemia. 
With  serum  from  the  last  disease  it  required  from  o.i  to  0.2  c.c. 


\ 


Warfie^'^*^^  T.  Longcope 


227 


j\  N.  sodium  hydrate  to  ne-'icralize  i  c.c.  of  serum.  In  the  sera 
from  three  patients  suffering  with  tuberculosis  there  was  only 
a  faint  cloud  and  no  acid.  Two  of  these  were  cases  of  acute 
tuberculous  pneumonia.  It  would  be  interesting  to  investigate 
this  subject  further.  No  acid,  and  very  faint  cloud  were  pro- 
duced in  beef  and  horse  serum.  Traces  of  acid  appeared  in 
calf  senmi,  while  in  normal  rabbit  senmi  the  reaction  was 
definitely  acid. 


TABLE  I. 

ACID  PRODUCTION   BY   GROWTH   OF   PXEUMOCOCCI   IN   PNEUMONIC   SERUM. 


Day  of  Disease  on  %vhich  Blood  -vvas  Drawn. 


Acid  Production 
after  4-6  Days' 
Growth  36.5°  C. 


2d..  . 
4th.  . 
6th.  . 
7th.  . 
9th.  . 
loth. 


Day  of  crisis 

4th  day  after  crisis 

5th     "'        "        "      

Undetermined  length  of  time,  empygema. 


+  +  + 

+  +  + 

+  +  + 

+  +  + 

+  +  + 


Besides  blood  serum,  pleural  and  spinal  exudates  were  ex- 
amined to  determine  if  in  these  fluids  acid  was  also  formed. 
One  cloudy  pleural  exudate  from  which  the  pneumococcus  was 
recovered  in  cultures  gave  a  moderate  acid  reaction  both  before 
and  after  growth  at  36.5°  C.  One  pleural  exudate  from  which 
streptococci  were  recovered  in  cultures  gave  no  acid  reaction 
even  after  several  days'  growth  at  36.5°  C.  The  spinal  fluid 
from  two  cases  of  meningitis  caused  by  the  pneumococcus  gave 
no  acid  reaction.  The  spinal  fluid  from  seven  cases  of  meningitis 
due  to  the  meningitis  coccus  gave  no  acid  reaction,  and  the 
spinal  fluid  from  one  case  of  tuberculous  meningitis  (tubercle 
bacilli  were  demonstrated  in  the  fluid)  gave  no  acid  reaction. 

There  is  then  some  substance  which  makes  its  appearance 
in  the  blood  serum  under  certain  conditions  and  from  Avhich  the 
pneumococcus  is  capable  of  forming  large  quantities  of  acid. 


228  Growth  of  Pneumococci  and  Streptococci 

This  substance  is  present  with  giaat  constancy  and  in  large 
amounts  during  an  attack  of  pneumonia,  but  seems  to  disappear 
rapidly  after  the  crisis.  It  may  be  present  in  general  strepto- 
coccus and  gonococcus  infections,  in  acute  articular  rheumatism, 
and  in  comparatively  small  amounts  in  uremia  and  chronic 
nephritis.  So  far  as  the  present  observations  go,  it  has  not  been 
demonstrated  in  cases  of  pulmonary  tuberculosis,  in  the  serum 
from  normal  individuals,  or  in  horse  or  beef  serum.  Traces 
eould  be  shown  to  exist  in  calf  and  rabbit  serum.  Apparently 
•the  substance  does  not  pass  readily  from  the  blood  into  exu- 
dates, for  only  small  quantities  of  acid  were  formed  in  the 
pleural  exudates  caused  by  the  pneumococcus,  and  none  in 
the  cerebro-spinal  exudate.  The  nature  of  the  substance  could 
not  be  discovered.  Almost  certainly  it  is  not  glucose,  for  the 
fresh  serum  did  not  give  the  reaction  for  this  sugar,  and  the 
large  quantity  of  acid  formed  precludes  the  idea  that  it  may 
have  arisen  from  traces  of  glucose.  It  has  been  shown  by  Hiss  ^ 
that  the  pneumococcus  is  capable  of  fermenting  a  long  list  of 
carbohydrates  including  the  starches,  such  as  inulin,  which  is 
not  acted  upon  by  streptococci.  He  suggests  further  that  gluco- 
proteids  and  nucleo-proteids  may  also  be  used  in  the  complicated 
metabolic  processes  of  the  pneumococcus. 

Rosenow  noted  that  if  pneumonic  serum  was  heated  to  56°  C. 
and  then  used  as  a  culture  medium  for  the  pneumococcus,  the 
amount  of  acid  formed  was  much  less  than  if  unheated  serum 
was  employed.  The  same  results  were  obtained  in  a  number  of 
our  cases.  If,  however,  the  serum  was  heated  to  56°-6o°  C.  for 
a  greater  length  of  time,  usually  one  or  two  hours,  the  growth  of 
the  pneumococcus  produced  firm  clotting  of  the  serum  within 
two  to  three  days.  This  coagulum  dissolved  on  the  addition  of 
alkalies.  The  same  experiment  was  repeated  several  times.  If 
by  the  growth  of  pneumococci  in  any  blood  serum,  acid  was  pro- 
duced in  such  amounts  that  it  required  from  o.2C.c.  too.6c.c.  of 
yV  N.  sodium  hydrate  to  neutralize  i  c.c.  of  serum,  heating  to 
57°-6o°  C.  produced  a  firm  white  coagulum.  This  occurred 
whether  the  serum  was  heated  before  the  growth  of  the  pneu- 

(•  Jotir.  of  Exper.  Med.,  1905,  vi,  317. 


Warfield  T.   Longcope 


229 


mococcus  or  afterwards.  Heating  of  the  serum  alone  to  57° 
produced  no  visible  change.  Heating  to  6g°-'j2°  C.  gave  rise  to 
a  firm  jelly-like,  clear  clot.  Without  heating,  the  growth  of 
pneumococci  produced  acid  but  gave  rise  to  no  coagulum. 
The  coagulum  was  always  soluble  in  an  excess  of  alkali.  When 
the  serum  was  neutralized  before  heating,  coagulation  did  not 
take  place  until  the  serum  was  heated  to  65°-7o°  C. 

Further  experiments  showed  that  the  addition  of  definite 
amounts  of  lactic  acid  to  several  different  sera  gave  rise  to  a 
coa'gulum  when  the  serum  was  heated  to  57°  C,  exactly  like  that 
obtained  with  the  serum  in  which  the  pneumococcus  had  grown 
and  produced  acid.  If,  however,  acid  was  added  below  a  certain 
limit  or  above  another  limit,  coagulation  did  not  take  place  on 
heating.  The  amount  of  acid  required  was  exactly  that  pro- 
duced in  the  pneumonic  sera  by  the  growth  of  the  pneumococcus, 
as  the  following  table  shows. 

TABLE  11. 


Serum. 


Amount  of  15  N.  So- 
diuiTi  Hydrate  Re- 
quired to  Neutralize 
I  c.c.  of  Serum  after 
Addition  of  Lac- 
tic Acid. 


Temperature  at 
which  Serum 
Coagulates. 


Beef 

Chronic  Nephritis  1 5 


72°  C 

57°   ' 
72^ 

57' 
57' 
65' 
57' 
65' 
75' 
70' 

57' 
57' 


The  fact  that  comparatively  large  quantities  of  acid  could  be 
added  to,  or  produced  in  pure  serum  without  the  coagulation  of 
the  serum,  suggested  that  the  clotting  of  the  serum-inulin  water 
of  Hiss  was  not  entirely  dependent  upon  the  acid  formed  by  the 
fermentation  of  the  carbohydrate. 

Beef  serum  was  used  in  the  preparation  of  the  latter  medium. 


230  Groioth  of  Pneumococci  and  Streptococci 

The  blood  was  carefully  collected  and  the  serum  kept  under 
sterile  conditions.  Before  making  up  the  medium,  specimens 
of  the  serum  were  incubated  at  37°  C.  to  make  sure  that  the  fluid 
was  not  contaminated.  To  this  unheated  serum  sufficient  quan- 
tities of  sterilized  inulin  water  were  added  to  give  the  final  mix- 
ture the  proportions  as  described  by  Hiss.  Pneumococci  grown 
in  this  unheated  serum-inulin  water  gave  rise  to  large  quantities 
of  acid,  biit  produced  no  coagulum.  The  medium ,  remained 
absolutely  fluid  though  a  little  turbid.  For  the  coagulation  of 
this  medium,  heating  at  least  above  57°  C.  is  absolutely  essential. 

That  blood  serum  when  diluted  with  two  parts  of  water  does 
not  coagulate  on  boiling  seems  to  be  unexplained.  But  this 
heating  must  have  some  effect  upon  the  serum.  The  fluid 
appears  almost  clear,  and  if  passed  through  a  Berkefeldt  filter 
becomes  absolutely  limpid.  Nevertheless,  this  serum  differs 
from  unheated  serum  in  the  fact  that  by  the  addition  of  small 
quantities  of  acids  coagulation  takes  place  immediately.  Even 
pure  albumen  may  be  sterilized  in  a  Koch  autoclave  without  fear 
of  coagulation,  provided  the  albumen  has  been  previously 
diluted  with  two  to  three  parts  of  water.  If,  however,  certain 
substances  are  added,  coagulation  takes  place  promptly.  At- 
tempts were  made  to  grow  pneumococci  in  pure  diluted  egg 
albumen.  Mixtures  of  one  part  of  egg  albumen  and  two  parts  of 
water  were  sterilized  at  100°  C.  The  mixture  remained  ab- 
solutely fluid  but  it  was  found  that  pneumococci  would  not 
thrive  in  the  medium.  Salt  and  peptone  were  added  to  the 
albumen  water,  but  it  was  impossible  to  sterilize  this  medium 
on  account  of  the  coagulation  which  took  place. 

It  seems  possible  therefore  that  when  serum  diluted  with 
water  is  heated  to  100°  C.  the  albumens  and  globulins  are  altered 
and  are  perhaps  present  in  a  so-called  colloidal  state.  In  this 
condition,  additions  of  small  amounts  of  acids  cause  rapid  pre- 
cipitation of  the  albumens  and  globulins  and  hence  produce 
coagulation  of  the  medium. 


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